Factor VIII mutation repair and tolerance induction

ABSTRACT

Methods of treating hemophilia A in a subject with an F8 gene mutation, wherein the F8 gene is repaired and the resultant repaired gene, upon expression, confers improved coagulation functionality to the encoded FVIII protein of the subject compared to the non-repaired F8 gene. The invention also includes methods of inducing immune tolerance to a FVIII replacement product ((r)FVIII) in a subject having a FVIII deficiency, wherein the F8 gene mutation is repaired and the repaired gene, upon expression, provides for the induction of immune tolerance to an administered replacement FVIII protein product. The invention also includes isolated nucleic acids, vectors, recombinant viruses, cells, and pharmaceutical compositions to repair the F8 gene.

STATEMENT OF RELATED APPLICATIONS

This application is the U.S. national stage entry of InternationalPatent Application No. PCT/US2013/073751 filed on Dec. 6, 2013, which,in turn, claims priority from Provisional U.S. Application No.61/734,678 filed Dec. 7, 2012, and Provisional U.S. Application No.61/888,424 filed Oct. 8, 2013. The entirety of U.S. ProvisionalApplication Nos. 61/734,678 and 61/888,424 are hereby incorporated byreference for all purposes.

GOVERNMENT INTEREST

This invention was made with government support under Grant NumberHL101851, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 22002-007WO1_Sequencelisting_patentin_ST25.txt.The text file is 109 kb, was created on Dec. 6, 2013, and is beingsubmitted electronically via EFS-Web.

BACKGROUND

Hemophilia (HA) is caused by loss-of-function mutations in the X-linkedFactor (F) VIII gene, F8. Infusion of replacement plasma-derived (pd) orrecombinant (r) FVIII is the standard of care to manage this chronicdisease. Infusion of replacement FVIII, however, is not a cure for HA.Spontaneous bleeding remains a serious problem especially for those withsevere HA, defined as circulating levels of FVIII coagulant activity(FVIII: C) below 1% of normal. Furthermore, the formation of anti-FVIIIantibodies occurs in about 20% of all patients and more often in certainsubpopulations of HA patients, such as African Americans (Viel K R,Ameri A, Abshire T C, et al. Inhibitors of factor VIII in black patientswith hemophilia. N Engl J Med. 360: 1618-27, 2009). Patients unable tobe treated with FVIII experience more painful, joint bleeding and overtime, a greater loss of mobility than patients whose HA is able to bemanaged with FVIII.

The approval in Europe of the first gene therapy obtained by uniQuire BVfor alipogenic tiparvovec (trade name Glybera) to treat lipoproteinlipase (LPL) deficiency is a milestone in the quest to bring gene-basedtherapeutics into clinical use. Tiparvovec (AAV1-LPL(S447X))incorporates an intact human LPL gene (LPL) variant, i.e. LPL(Ser447X),in an adeno-associated virus (AAV) vector, which is deliveredintramuscularly (Gaudet D, Méthot J, Déry S, et al. Efficacy andlong-term safety of alipogene tiparvovec (AAV1LPL(S447X)) gene therapyfor lipoprotein lipase deficiency: an open-label trial. Gene Ther. Jun.21, 2012. [Epub ahead of print]). The benefits of restoring continuouscirculation of clinically meaningful levels of FVIII has motivatedintense efforts over decades to develop an effective gene therapy forhemophilia. Use of an AAV vector to deliver the gene that encodes FIX isbearing fruit in the treatment of hemophilia B (HB) (Nathwani A,Tuddenham E G D, Rangarjan S, et al. Adeno-associated viral vectormediated gene transfer for hemophilia B. Blood. 118(21): 4-5, 2011). HBin six adult patients has been converted from a severe form to mild ormoderate HB, following intravenous infusion of an AAV8 vectorincorporating human F9 under the control of a liver restricted promoter(High K A. The gene therapy journey for hemophilia: are we there yet?Blood. 120(23): 4482-7, 2012). These patients have maintained acirculating FIX coagulant activity (FIX: C) level ranging from 1-6% ofnormal for 3 years.

Although very encouraging, the AAV vector is not suitable for many HBpatients and safety concerns remain. AAV vectors have been engineeredfrom a wild-type parvovirus capable of naturally infecting humans(Calcedo R, Morizono H, Wang L, et al. Adeno-associated virus antibodyprofiles in newborns, children, and adolescents. Clin Vaccine Immunol.18(9): 1586-8, 2011; High K A. The gene therapy journey for hemophilia:are we there yet? Blood. 120(23): 4482-7, 2012). In the current AAVtrial, patients are screened for neutralizing antibodies against AAV.Thus, about 30-50% of hemophilia patients may not be eligible for thistreatment (High K A. The gene therapy journey for hemophilia: are wethere yet? Blood. 120(23): 4482-7, 2012). Patients with liver diseaseare also not eligible for this therapy pending a better understanding ofsafety. Furthermore, because the vectors are predominantlynon-integrating, they are not suitable for use with young patientsbecause expression of FIX would be expected to be lost as the patientgrows (High K A. The gene therapy journey for hemophilia: are we thereyet? Blood. 120(23): 4482-7, 2012).

Transmission of AAV to the germ-line (semen) has been reported, but thisappears to be transient (High K A. The gene therapy journey forhemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). There is atrend toward a dose-dependent memory T-cell mediated response to AAV.This was seen in the first HB trial done with AAV and again in thecurrent trial for HB (High K A. The gene therapy journey for hemophilia:are we there yet? Blood. 120(23): 4482-7, 2012). Whereas dosing in fourpatients in the range of 2×10¹¹ vg/kg did not provoke a T-cell response,it provided only low circulating FIX: C levels (1-3%). Dosing at 2×10¹²vg/kg in two patients led to initial robust levels of FIX incirculation, 8-10%; but, at 8-weeks post infusion, FIX levels began tofall and patients' liver enzymes rose (High K A. The gene therapyjourney for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012).Treatment with prednisolone was effective in restoring normal liverenzyme levels and reducing AAV-capsid specific T-cells within PBMCs(High K A. The gene therapy journey for hemophilia: are we there yet?Blood. 120(23): 4482-7, 2012). In one patient, FIX fell to 2% of normal,but the other maintained FIX levels at 6% of normal (High K A. The genetherapy journey for hemophilia: are we there yet? Blood. 120(23):4482-7, 2012). Questions remain about the long-term safety of therapywith AAV. Sequencing studies of organs from animals treated with AAVdemonstrate that integration of AAV occurs (Naki H, Yant S R, Storm T A,Fuess S, Meuse L, Kay M S. Extrachromosomal recombinant adenoassociatedvirus vector genomes are primarily responsible for stable livertransduction in vivo. J Virol. 75(15): 6969-76, 2001). There is also onereport of neonatal mice injected with AAV2 experiencing an increase inhepatocellular carcinoma with some tumors containing vector DNA (Chuah MK, Nair N, VandenDriessche T. Recent progress in gene therapy forhemophilia. Hum Gene Ther. 23(6): 557-65, 2012). Whether AAV will proveto have any utility for delivering F8 to HA patients is an openquestion. The cloning capacity of the vector is limited to replacementof the virus' 4.8 kilobase genome. Promising preclinical results havebeen obtained using an AAV packaged, non-naturally occurring F8 cDNA,encoding B-domain-deleted (BDD)-rFVIII. One major caveat is that thedosing required to achieve desired levels of circulating FVIII (1-7.8%)in these studies was a log higher than the highest dose used in thecurrent HB trial (Chuah M K, Nair N, VandenDriessche T. Recent progressin gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012;Jonathan D. Finn, Margareth C. Eradication of neutralizing antibodies toFVIII in canine hemophilia A after liver gene therapy. Blood 116:5842-5848 2010). Experience in the HB trial suggests that a memoryT-cell response targeting liver cells would occur at these doses (High KA. The gene therapy journey for hemophilia: are we there yet? Blood.120(23): 4482-7, 2012; Chuah M K, Nair N, VandenDriessche T. Recentprogress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65,2012).

A substantial effort has been made to develop lentiviral vectors for thedelivery of clotting factors (Chuah M K, Nair N, VandenDriessche T.Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6):557-65, 2012). Lentiviral vectors can transfect non-cycling hepatocytesand integrate into the host genome. While the latter attribute mayafford long term expression of the factor, it raises serious safetyconcerns regarding insertional mutagenesis leading to activation ofoncogenes or inactivation of repressors. Studies in an HB mouse indicatethat a preferred lentiviral vector could be one that harbors aninactivation mutation of the integrase, termed an IDLV (integrasedefective lentiviral viral vector) (Matria J, Chuah M K, VandenDriesscheT. Recent advances in lentiviral vector development and applications.Mol Ther. 18: 477-90, 2010). Although factor expression is diminishedfollowing the elimination of integration, it appears this may be offsetin the case of FIX by use of a rare hyper-activating variant in acodon-optimized F9 cDNA which dramatically boosts circulating FIX levels(Matria J, Chuah M K, VandenDriessche T. Recent advances in lentiviralvector development and applications. Mol Ther. 18: 477-90, 2010).Another obstacle with lentivirus is its competence to transfect APCswhich triggers deleterious T-cell mediated immune responses that canneutralize the secreted clotting factor; or eliminate the transducedhepatocytes (VandenDriessche T, Thorerezn L, Naldini L, et al.Lentiviral vectors containing the human immunodeficiency virus type-1central polypurine track can efficiently transduce non-dividinghepatocytes and antigen presenting cells in vivo. Blood. 100: 813-22,2002). This can be reduced in animal models by including an additionallayer of post-transcriptional control, mediated by the use of endogenousmicroRNA (miR) (Matria J, Chuah M K, VandenDriessche T. Recent advancesin lentiviral vector development and applications. Mol Ther. 18: 477-90,2010). Whether manipulations designed to mute the immune response tolentivirus will translate to man is unknown. In contrast to what occursin man, in the canine model, AAV vectors did not elicit an immuneresponse. Hence, while animal models have proven predictive with regardto forecasting delivery of factor clotting activity; no animal model,including the canine, recapitulates the more refined and sophisticatedhuman immune response.

Use of autologous cells engineered with viral elements or nucleasescapable of genomic editing may permit greater safety than intravenousdelivery of targeted virus. Ex vivo protocols allow for screening of thegenomes of manipulated cells to assess the frequency or viralinsertions, double strand breaks in DNA (DSBs) or other potentiallymutagenic events (Li H, Haurigot V, Doyon Y, et al. In vivo genomeediting restores haemostasis in a mouse model of haemophilia. Nature.475(7355): 217-21, 2011). Levels of blood clotting proteins needed tomaintain hemostasis may be more readily achieved by expansion of largepopulations of cells ex vivo and reintroduction(s) into the patient.Promising work has been done with murine hematopoietic stem cells (HSCs)transduced with lentivirus to express FVIII, including in HA mice withhigh-titer FVIII inhibitors (Calcedo R, Morizono H, Wang L, et al.Adeno-associated virus antibody profiles in newborns, children, andadolescents. Clin Vaccine Immunol. 18(9): 1586-8, 2011; Chuah M K, NairN, VandenDriessche T. Recent progress in gene therapy for hemophilia.Hum Gene Ther. 23(6): 557-65, 2012). Enthusiasm for these approaches istempered, however, by recognition that in order to promote engraftment,conditioning agents such as busulfan, which can have serious sideeffects are required (Chuah M K, Nair N, VandenDriessche T. Recentprogress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65,2012). Furthermore, concerns remain regarding the long-term consequencesof lentiviral incorporation into the genomes of hematopoietic stem cells(HSCs).

In addition, there is a critical need to identify ways to avoid FVIIIinhibitor development and to abate a FVIII inhibitor response. Anarduous (frequent infusions of FVIII) and extremely expensive (˜$1MM/patient) protocol called Immune Tolerance Induction (ITI) is the onlyapproach proven to eradicate FVIII inhibitors in HA patients, yet failsamong 30% of inhibitor patients (Morfin M. et. al. European study onorthopaedic status of haemophilia patients with inhibitors. HaemophiiaSep; 13(5):606-12 2007). Twenty percent of HA patients with the F8I22Idevelop inhibitors. In addition, HA patients with missense mutationsexpressed in the C2 domain of FVIII are more prone to inhibitorformation than those with mutations expressed elsewhere in theirendogenous FVIII (8.7% C1/C2 domain vs. 3.6% non-C1/C2-domain; p-value:0.01 sample size 1135 HA patients).

SUMMARY

The invention includes a method of treating a human subject withhemophilia A comprising selectively targeting and replacing a portion ofthe subject's genomic F8 gene sequence containing a mutation in the genewith a replacement sequence. In one embodiment, the resultant repairedgene, upon expression, confers improved coagulation functionality to theencoded FVIII protein of the subject compared to the non-repaired F8gene. In one embodiment, the repaired gene, upon expression, providesfor the induction of immune tolerance to an administered replacementFVIII protein product.

In one aspect of the invention, a method of treating hemophilia A in asubject is provided comprising introducing into a cell of the subjectone or more isolated nucleic acids encoding a nuclease that targets aportion of the F8 gene containing a mutation that causes hemophilia A,wherein the nuclease creates a double stranded break in the F8 gene; andan isolated nucleic acid comprising a donor sequence comprising (i) anucleic acid encoding a truncated FVIII polypeptide or (ii) a native F83′ splice acceptor site operably linked to a nucleic acid encoding atruncated FVIII polypeptide, wherein the nucleic acid comprising the (i)a nucleic acid encoding a truncated FVIII polypeptide or (ii) native F83′ splice acceptor site operably linked to a nucleic acid encoding atruncated FVIII polypeptide is flanked by nucleic acid sequenceshomologous to the nucleic acid sequences upstream and downstream of thedouble stranded break in the DNA, and wherein the resultant repairedgene, upon expression, confers improved coagulation functionality to theencoded FVIII protein of the subject compared to the non-repaired F8gene. In one particular embodiment, the donor sequence comprises anative F8 3′ splice acceptor site operably linked to a nucleic acidencoding a truncated FVIII polypeptide. In one embodiment, the subjectis a human. In one embodiment, the nucleic acids encoding the nucleaseintroduced into the cell are ribonucleic acids.

In one aspect, the invention provides a method of inducing immunetolerance to a FVIII replacement product ((r)FVIII) in a subject havinga FVIII deficiency and who will be administered, is being administered,or has been administered a (r)FVIII product comprising introducing intoa cell of the subject one or more nucleic acids encoding a nuclease thattargets a portion of the F8 gene containing a mutation that causeshemophilia A, wherein the nuclease creates a double stranded break inthe F8 gene; and an isolated nucleic acid comprising a donor sequencecomprising a (i) a nucleic acid encoding a truncated FVIII polypeptideor (ii) a native F8 3′ splice acceptor site operably linked to a nucleicacid encoding a truncated FVIII polypeptide, wherein the nucleic acidcomprising the (i) a nucleic acid encoding a truncated FVIII polypeptideor (ii) native F8 3′ splice acceptor site operably linked to a nucleicacid encoding a truncated FVIII polypeptide is flanked by nucleic acidsequences homologous to the nucleic acid sequences upstream anddownstream of the double stranded break in the DNA, and wherein therepaired gene, upon expression, provides for the induction of immunetolerance to an administered replacement FVIII protein product. In oneparticular embodiment, the donor sequence comprises a native F8 3′splice acceptor site operably linked to a nucleic acid encoding atruncated FVIII polypeptide. In one embodiment, the truncated FVIIIpolypeptide amino acid sequence shares homology with a positionallycoordinated portion of the (r)FVIII amino acid sequence. In oneembodiment, the truncated FVIII polypeptide amino acid sequence sharescomplete homology with a positionally correlated portion of the (r)FVIIIamino acid sequence. In one embodiment, the subject is a human. In oneembodiment, the nucleic acids encoding the nuclease introduced into thecell are ribonucleic acids.

In one embodiment, the F8 mutation targeted for replacement is a pointmutation, a deletion, or an inversion. In one embodiment of the abovedescribed aspects, the nuclease targets the F8 gene at intron 1. In oneembodiment, the nuclease targets a F8 gene at intron 14. In oneembodiment, the nuclease targets the F8 gene at intron 22. In aparticular embodiment, the mutation targeted for repair is an intron 22inversion (I22I).

In one embodiment of the above described aspects, the nuclease targetsthe F8 gene at exon1/intron 1 junction, exon14/intron 14 junction, orexon22/intron 22 junction.

In one embodiment of the above described aspects, the encoded nucleaseis a zinc finger nuclease (ZFN). In one embodiment, the encoded nucleaseis a Transcription Activator-Like Effector Nuclease (TALEN). In oneembodiment, the encoded nuclease is a CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats)-associated (Cas) nuclease. In oneparticular embodiment, the encoded nuclease is a TALEN.

In the methods provided herein, the nucleic acids can optionally be in avector.

In one embodiment of the above described aspects, the isolated nucleicacids described above can be administered directly to the subject sothat introduction into the subject's cell occurs in vivo. The isolatednucleic acids can be delivered to the subject's cells in vivo by avariety of mechanisms, including through uptake of naked DNA, liposomefusion, or through viral transduction, for example adenovirus,adeno-associated virus (AAV), or lentivirus introduction, as describedfurther below.

In one embodiment of the above described aspects, the isolated nucleicacids described above can be administered ex vivo to a cell that hasbeen isolated from the subject. The isolated nucleic acids can bedelivered to the cells via any gene transfer mechanism, for example,calcium phosphate mediated gene delivery, electroporation,microinjection, liposome delivery, endocytosis, or viral delivery, forexample, adenoviral, AAV, or lentiviral delivery, as described furtherbelow. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject. In one embodiment, the nucleic acids encoding thenuclease can be introduced into the cell as ribonucleic acids, forexample mRNA. Alternatively, the nucleic acids can optionally be in aDNA vector.

In one embodiment of the above described aspects, the cells areendothelial cells. In one embodiment, the endothelial cell is a bloodoutgrowth endothelial cells (BOECs). In one embodiment, the cellsrepaired reside within the liver. In one embodiment, the cells arehepatocytes. In one embodiment, the cells are liver sinusoidalendothelial cells (LESCs). In one embodiment, the cells are stem cells.In one embodiment, the stem cells are induced pluripotent stem cells(iPSCs).

In one embodiment of the above described aspects, the nucleic acidsdescribed above are introduced into blood outgrowth endothelial cells(BOECs) that have been co-cultured with additional cell types. In oneembodiment, the cells are blood outgrowth endothelial cells (BOECs) thathave been co-cultured with hepatocytes or liver sinusoidal endothelialcell (LESCs), or both. In one embodiment, the cells are blood outgrowthendothelial cells (BOECs) that have been co-cultured with inducedpluripotent stem cells (iPSCs).

Cells comprising the nucleic acids set forth herein are furtherprovided. These cells can be, for example, endothelial cells, LSECs,BOECs, stem cells, for example iPSCs, or hepatocytes.

Recombinant viruses comprising any of the nucleic acids set forth hereinare also provided.

Pharmaceutical compositions comprising any of the nucleic acids, vectorscomprising the nucleic acids, recombinant viruses comprising the nucleicacids, or cells comprising the nucleic acids described herein are alsoprovided. These pharmaceutical compositions can be in a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is schematic illustration of the wild-type and intron-22-invertedFVIII loci (F8 & F8_(I22I)) and their expressed protein products(FVIII_(FL) & FVIII_(B) for F8 and FVIII_(I22I) & FVIII_(B) forF8_(I22I)).

FIG. 2 is a schematic illustration of a TALEN-mediated genomic editingthat can be used to repair the human intron-22 (I22)-inverted F8 locus,F8_(I22I).

FIG. 3 shows a functional heterodimeric TALEN, comprised of its left andright monomer subunits (TALEN-L and TALEN-R), targeting the human F8gene.

FIG. 4 shows a functional heterodimeric TALEN, comprised of its left andright monomer subunits (TALEN-L and TALEN-R) targeting the canine F8gene

FIG. 5 illustrates the TALEN approach linking Exon 22 of the F8 gene toa nucleic acid encoding a truncated FVIII polypeptide encoding exons23-26.

FIG. 6 illustrates the TALEN approach linking Intron 22 to a F8 3′splice acceptor site operably linked to a nucleic acid encoding atruncated FVIII polypeptide.

FIG. 7 shows a comparison of expected genomic DNA, spliced RNA andproteins pre and post repair.

FIG. 8 shows PCR primer design to confirm correct integration of exons23-26 to repair the human intron-22 (I22)-inverted F8 locus, F8_(I22I).

FIG. 9 illustrates the donor plasmid targeting the F8 Exon22/Intron22junction using a TALEN nuclease, zinc finger nuclease, or cas nucleaseapproach.

FIG. 10 illustrates the donor plasmid targeting the F8 Exon1/Intron1junction using a TALEN nuclease, zinc finger nuclease, or cas nucleaseapproach.

FIG. 11 illustrates the donor plasmid targeting the F8 Intron 22 regionusing a TALEN nuclease, zinc finger nuclease, or cas nuclease approach.

FIG. 12 illustrates the donor plasmid targeting the F8 Intron 1 regionusing a TALEN nuclease, zinc finger nuclease, or cas nuclease approach.

FIG. 13 illustrates the CRISPR/Cas9-mediated F8 repair strategytargeting intron 1.

DETAILED DESCRIPTION

The invention includes a method of treating a subject with hemophilia Acomprising selectively targeting and replacing a portion of thesubject's genomic F8 gene sequence containing a mutation in the genewith a replacement sequence. In one embodiment, the resultant repairedgene containing the replacement sequence, upon expression, confersimproved coagulation functionality to the encoded FVIII protein of thesubject compared to the non-repaired F8 gene. Preferably, the levels offunctional FVIII in circulation are adequate to obviate or reduce theneed for infusions of replacement FVIII in the subject. In oneembodiment, expression of functional FVIII reduces whole blood clottingtime (WBCT). In one embodiment, the repaired gene, upon expression,provides for the induction of immune tolerance to an administeredreplacement FVIII protein product. In one embodiment, the subject is ahuman.

In one aspect of the invention, a method of treating hemophilia A in asubject is provided comprising introducing into a cell of the subjectone or more nucleic acids encoding a nuclease that targets a portion ofthe human F8 gene containing a mutation that causes hemophilia A,wherein the nuclease creates a double stranded break in the F8 gene; andan isolated nucleic acid comprising (i) a nucleic acid encoding atruncated FVIII polypeptide or (ii) a native F8 3′ splice acceptor siteoperably linked to a nucleic acid encoding a truncated FVIIIpolypeptide, wherein the nucleic acid comprising the (i) a nucleic acidencoding a truncated FVIII polypeptide or (ii) native F8 3′ spliceacceptor site operably linked to a nucleic acid encoding a truncatedFVIII polypeptide is flanked by nucleic acid sequences homologous to thenucleic acid sequences upstream and downstream of the double strandedbreak in the DNA, and wherein the resultant repaired gene, uponexpression, confers improved coagulation functionality to the encodedFVIII protein of the subject compared to the non-repaired F8 gene.

In one aspect, the invention is a method of inducing immune tolerance toa FVIII replacement product (r)FVIII in a subject having a FVIIIdeficiency and who will be administered, is being administered, or hasbeen administered a (r)FVIII product comprising introducing into a cellof the subject one or more nucleic acids encoding a nuclease thattargets a portion of the F8 gene containing a mutation that causeshemophilia A, wherein the nuclease creates a double stranded break inthe F8 gene; and an isolated nucleic acid comprising (i) a nucleic acidencoding a truncated FVIII polypeptide or (ii) a native F8 3′ spliceacceptor site operably linked to a nucleic acid encoding a truncatedFVIII polypeptide, wherein the nucleic acid comprising the (i) a nucleicacid encoding a truncated FVIII polypeptide or (ii) native F8 3′ spliceacceptor site operably linked to a nucleic acid encoding a truncatedFVIII polypeptide is flanked by nucleic acid sequences homologous to thenucleic acid sequences upstream and downstream of the double strandedbreak in the DNA, and wherein the repaired gene, upon expression,provides for the induction of immune tolerance to an administeredreplacement FVIII protein product. The person administered the cells mayhave no anti-FVIII antibodies or have anti-FVIII antibodies as detectedby ELISA or Bethesda assays. In one embodiment, the truncated FVIIIpolypeptide amino acid sequence shares homology with a portion of the(r)FVIII amino acid sequence. In one embodiment, the truncated FVIIIpolypeptide amino acid sequence shares homology with a similar portionof the (r)FVIII amino acid sequence. In one embodiment, the truncatedFVIII polypeptide amino acid sequence shares complete homology with asimilar portion of the (r)FVIII amino acid sequence.

1. Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids. As used throughout, by subject is meantan individual. Preferably, the subject is a mammal such as a primate,and, more preferably, a human. Non-human primates are subjects as well.The term subject includes domesticated animals, such as cats, dogs,etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.)and laboratory animals (for example, ferret, chinchilla, mouse, rabbit,rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medicalformulations are contemplated herein.

The term “truncated FVIII polypeptide” refers to an amino acid sequencethat contains less than the full length FVIII protein. The truncatedFVIII polypeptide can be truncated from the 5′ end of the amino acidsequence. This produces an amino acid sequence corresponding to aportion of the FVIII protein, where a variable amount of the amino acidsequence is missing from the 5′ end of the protein. In one embodiment,the truncated FVIII polypeptide contains exons 23-26. In one embodiment,the truncated FVIII polypeptide contains exons 2-26. In one embodiment,the truncated FVIII polypeptide contains exons 15-26.

2. F8 Gene Mutations

The F8 gene, located on the X chromosome, encodes a coagulation factor(Factor VIII) involved in the coagulation cascade that leads toclotting. Factor VIII is chiefly made by cells in the liver, andcirculates in the bloodstream in an inactive form, bound to vonWillebrand factor. Upon injury, FVIII is activated. The activatedprotein (FVIIIa) interacts with coagulation factor IX, leading toclotting.

Mutations in the F8 gene cause hemophilia A (HA). Over 2100 mutations inthe gene have been identified, including point mutations, deletions, andinsertion. One of the most common mutations includes inversion of intron22, which leads to a severe type of HA. Mutations in F8 can lead to theproduction of an abnormally functioning FVIII protein or a reduced orabsent amount of circulating FVIII protein, leading to the reduction ofor absence of the ability to clot in response to injury.

In one aspect, the present invention is directed to the targeting andrepair of F8 gene mutations in a subject suffering from hemophilia Ausing the methods described herein. Approximately 98% of patients with adiagnosis of hemophilia A are found to have a mutation in the F8 gene(i.e., intron 1 and 22 inversions, point mutations, insertions, anddeletions).

Identification of an HA subject's mutation for targeting and repair canbe readily made by using techniques known in the art. For example, DNAfrom the subject can be extracted from leukocytes in whole blood and allthe endogenous coding regions and splice junctions of the F8 gene can beanalyzed by restriction analysis, direct DNA sequence analysis,Denaturing Gradient Gel Electrophoresis (DGGE), Chemical MismatchCleavage (CMC), and Denaturing High Performance Liquid Chromatography(DHPLC) (see, for example: Higuchi et al., Characterization of mutationsin the factor VIII gene by direct sequencing of amplified genomic DNA.Genomics 1990: 6(1); 65-71, Schwaab et al. Mutations in hemophilia A. BrJ Haematol 1993; 83: 450-458; Schwaab et al. Factor VIII gene mutationsfound by a comparative study of SSCP, DGGE, and CMC and their analysison a molecular model of factor VIII protein. Hum Genet 1997; 101:323-332; Oldenburg et al. Evaluation of DHPLC in the analysis ofhemophilia A. J Biochem Biophys Methods 2001; 47: 39-51). Intron 22inversions account for approximately 45% and intron 1 inversions accountfor approximately 2% to 3%, of mutations associated with severehemophilia A. The identification of inversions is well known in the art(see, for example, Viel at al. Inhibitors of Factor VIII in BlackPatients with Hemophilia. N Engl J Med 2009; 360(16): 1618-1627).

In one embodiment of the present invention, the gene mutation targetedfor repair is a point mutation. In one embodiment, the gene mutationtargeted for repair is a deletion. In one embodiment, the gene mutationtargeted for repair is an inversion. In one embodiment, the genemutation targeted for repair is an inversion of intron 1. In oneembodiment, the gene mutation targeted for repair is an inversion ofintron 22.

The intron 22 inversion mutation of the F8 gene accounts for ˜45% ofsevere haemophilia A and is caused by an intra-chromosomal recombinationwithin the gene. FIG. 1 shows a schematic illustration of the wild-typeand intron 22-inverted FVIII loci (F8 & F8I22I). Transcription from theF8 promoter of both the F8 (wild-type) & F8I22I loci, which is normallyfunctioning in both forms, yields polyadenylated mRNAs. The F8 mRNA has26 exons, E1-E22 and E23-E26, all of which encode the amino acids foundin the FVIII protein. Conversely, the F8I22I mRNA has at least 24 exons,E1-E22 (they are the same in F8 and thus encode FVIII amino acidsequence), and E23C & E24C (they are cryptic and encode no FVIII aminoacid sequence). The sequence of intron-22, in both F8 & F8I22I, containsa bi-directional promoter that transcribes two additional mRNAs from thetwo genes: F8A, which is oriented oppositely to that of F8 & F8I22I andcontains a single exon (box designated E1A), and F8B, which containsfive exons that are oriented similarly transcriptionally to that of F8 &F8I22I and contains a single non-F8 first exon within I22 (boxdesignated E1B) followed by four additional exons, which are identicalto E23-E26 of F8. The F8A mRNA encodes the FVIIIA protein, which is nowknown as HAP40 (a cytoskeleton-interacting protein involved inendocytosis and thus functionally unrelated to the coagulation system)and has no FVIII amino acid sequence. The F8B mRNA encodes FVIII B, aprotein with unknown function that has 8 non-FVIII amino acid residuesat its N-terminus followed by 208 residues that represent FVIII residues2125-2332.

3. Targeting Nucleases

The present invention provides for the targeting and repair of a mutatedF8 gene in a subject with HA, including by introducing into a subject'scell one or more nucleic acids encoding a nuclease that specificallytargets the F8 mutation. As discussed above, each subject's HA mutationfor targeting and repair can be readily determined using techniquesknown in the art. The identified mutation in the subject can then bedirectly targeted by nucleases for correction. Alternatively, thesubject's HA mutations can be corrected by targeting a region of the F8gene upstream (or 5′) from where the mutation occurs, and adding backthe corresponding downstream coding regions of the F8 gene. For example,intron 14 could be targeted by the nucleases. This allows for generepair of downstream mutations (i.e. missense mutations in exon 15 toexon 26) and inversions (such as the intron 22 inversion), due to thereplacement of exons 15 to 26 with the wild-type sequences. In otherembodiments, the F8 gene can be targeted at additional regions upstream,in order to capture an increasing proportion of F8 gene mutationscausing HA. Thus, the nucleases can be engineered to specifically targeta subject's F8 mutation, or alternatively, may target regions upstreamof a subject's F8 mutation, in order to correct the mutation.

In the methods and compositions set forth herein, the one or morenucleic acids encoding a nuclease that targets a mutation in F8 forrepair, for example, an intron 22 mutation in human F8, can be, forexample, a transcription activator-like effector nuclease (TALEN), azinc finger nuclease (ZFN), or a CRISPR (Clustered Regularly InterspacedShort Palindromic Repeats)-associated (Cas) nuclease.

Transcription Activator-Like Effector Nucleases (TALENs)

Transcription Activator-Like Effector Nucleases (TALENs) are emerging asa preferred alternative to zinc finger nucleases (ZFNs) for certaintypes of genome editing. The C-terminus of the TALEN component carriesnuclear localization signals (NLSs), allowing import of the protein tothe nucleus. Downstream of the NLSs, an acidic activation domain (AD) isalso present, which is probably involved in the recruitment of the hosttranscriptional machinery. The central region harbors the mostfascinating feature of TALENs and the most versatile. It is made up of aseries of nearly identical 34/35 amino acids modules repeated in tandem.Residues in positions 12 and 13 are highly variable and are referred toas repeat-variable di-residues (RVDs). Studies of TALENs such as AvrBs3from X. axonopodis pv. vesicatoria and the genomic regions (e.g.,promoters) they bind, led two teams to “crack the TALE code” byrecognizing that each RVD in a repeat of a particular TALE determinesthe interaction with a single nucleotide. Most of the variation betweenTALEs relies on the number (ranging from 5.5 to 33.5) and/or the orderof the quasi-identical repeats. Estimates using design criteria derivedfrom the features of naturally occurring TALEs suggest that, on average,a suitable TALEN target site may be found every 35 base pairs in genomicDNA. Compared with ZFNs, the cloning process of TALENs is easier, thespecificity of recognized target sequences is higher, and off-targeteffects are lower. In one study, TALENs designed to target CCR5 wereshown to have very little activity at the highly homologous CCR2 locus,as compared with CCR5-specific ZFNs that had similar activity at the twosites.

FIGS. 2 and 3 are exemplary illustrations outlining the use of a nucleicacid encoding a TALEN nuclease that can be used to repair the F8 genein, for example, a human with an intron-22 (I22)-inverted F8 locus,F8I22I. As illustrated in FIG. 2(A), the major transcription unit of theF8I22I locus consists of 24 exons, which are designated exons 1-22 andexons 23C & 24C. The first 22 are the same as exons 1-22 of thewild-type FVIII structural locus (F8) but the last two (exon-23C &exon-24C) are cryptic and non-functional in non-hemophilic individualsas well as in patients whose HA is caused by F8 gene abnormalities otherthan the I22I-mutation. As illustrated in FIG. 2(B) the strategy torepair the I22I-mutation consists of introducing in the cell of thesubject a nucleic acid encoding a functional TALEN—which is aheterodimeric nuclease comprised of a monomer subunit that binds 5′ ofthe desired genome editing site (TALEN-L) and one that binds 3′ of it(TALEN-R)—that is specific for a DNA sequence that is present in only asingle copy per haploid human genome, which is approximately 1 kbdownstream of the 3′-end of exon-22. Upon expression, once both monomersare bound to this specific sequence, their individual Fok1 nucleasedomains dimerize to form the active enzyme that catalyzes adouble-stranded (ds) break in the DNA between their binding sites. If ads-DNA break occurs in the presence of a second nucleic acid, forexample a nucleic acid comprising a native FVIII 3′ splice acceptor siteoperably linked to a nucleic acid encoding a truncated FVIII polypeptideencoding exons 23-26 (i.e., a “donor plasmid (DP)”), which contains astretch of DNA with a left homology (HL) arm and right homology (HL) armthat have identical DNA sequences to that in the native chromosomal DNA5′ and 3′ of the region flanking the break-point, homologousrecombination (HR) occurs very efficiently. Following HR, the DNAsegment between the left and right homology arms (which in this casecontains a partial human F8 cDNA that contains, in-frame, all of exons23-25 and the coding sequence of exon-26, with a functional 3′-splicesite at its 5′-end) becomes permanently ligated/inserted into thechromosome. Since the DNA segment between the left and right homologyarms comprises a partial human F8 cDNA (which, as shown in FIG. 2,contains, in-frame, all of exons 23-25 and the coding sequence ofexon-26) fused at its 5′-end to a functional 3′-splice site, this TALENcatalyzes repair and converts F8I22I into wild-type F8-like locus andrestore its ability to drive synthesis of a full-length fully functionalwild-type FVIII protein.

FIG. 3 shows a functional heterodimeric TALEN, comprised of left andright monomer subunits (TALEN-L and TALEN-R), bound to its target“editing” sequence in intron 22 (I22) of the human FVIII structurallocus (F8), ˜1 kb downstream of the 3′-end of exon-22 (FIG. 3). Becausethe target binding sequence of each monomer is the same in both awild-type FVIII gene (F8) and an I22-inverted FVIII gene (F8I22I), thisTALEN edits each locus equally well. Following binding of this TALEN'smonomeric subunits to their target I22-sequences in the F8I22I locus ofa patient with severe HA caused by the I22-inversion (I22I)-mutation,the individual Fok1 nuclease domains are able to form a homo-dimer, i.e.the active form of the enzyme, which catalyzes a double-stranded (ds)break in the DNA between the monomer binding sites. If a ds-DNA breakoccurs in the presence of a donor plasmid containing the replacement or“repairing” sequence, which contains a stretch of DNA with left andright arms that have identical DNA sequences to that in the nativechromosomal DNA, in the region flanking the break-point (see FIG. 2),homologous recombination (HR) occurs very efficiently. Following HR, theDNA segment between the left and right homology arms (which, as shown inFIG. 2, contains a partial human F8 cDNA that contains, in-frame, all ofexons 23-25 and the coding sequence of exon-26, with a functional3′-splice site at its 5′-end—i.e., the repairing sequence) becomespermanently ligated/inserted into the chromosome. Because the DNAsegment between the left and right homology arms comprises a partialhuman F8 cDNA (which, as shown in FIG. 2, contains, in-frame, all ofexons 23-25 and the coding sequence of exon-26) fused at its 5′-end to afunctional 3′-splice site, this TALEN catalyzes repair and convertsF8I22I into wild-type F8-like locus and restore its ability to drivesynthesis of a full-length fully functional wild-type FVIII protein.

Likewise, FIG. 4 shows a functional heterodimeric TALEN targeting a F8mutation in canine, comprised of its left and right monomer subunits(TALEN-L and TALEN-R), bound to its target “editing” sequence in theintron-22 (I22) of the canine FVIII structural locus (cF8), ˜0.25 kbdownstream of the 3′-end of exon-22. Because the target binding sequenceof each monomer is the same in both a wild-type canine FVIII gene (cF8)and an I22-inverted FVIII gene (cF8I22I), this TALEN edits each locusequally well. Following binding of this TALEN's monomeric subunits totheir target I22-sequences in the cF8I22I locus of a dog with severe HAcaused by the I22-inversion (I22I)-mutation, their individual Fok1nuclease domains are able to form a homo-dimer, i.e. the active form ofthe enzyme, which catalyzes a double-stranded (ds) break in the DNAbetween the monomer binding sites. If a ds-DNA break occurs in thepresence of a donor plasmid, which contains a stretch of DNA with leftand right arms that have identical DNA sequences to that in the nativechromosomal DNA, in the region flanking the break-point (see FIG. 3 forthe human F8 locus), homologous recombination (HR) occurs veryefficiently. Following HR, the DNA segment between the left and righthomology arms (which contains a partial cF8 cDNA that contains,in-frame, all of exons 23-25 and the coding sequence of exon-26, with afunctional 3′-splice site at its 5′-end) becomes permanentlyligated/inserted into the canine X-chromosome. Because the DNA segmentbetween the left and right homology arms comprises a partial cF8 cDNA(which, as shown in FIG. 2 for the human F8I22I, contains, in-frame, allof canine exons 23-25 and the coding sequence of canine exon-26) fusedat its 5′-end to a functional 3′-splice site, this TALEN catalyzesrepair and converts cF8I22I into a wild-type cF8-like locus thatrestores its ability to drive synthesis of a full-length fullyfunctional wild-type canine FVIII protein.

FIG. 5 illustrates a TALEN-mediated strategies to repair the humanFactor VIII (FVIII) gene (F8) mutations in >50% of all patients withsevere hemophilia-A (HA), including the highly recurrent intron-22(I22)-inversion (I22I)-mutation. FIG. 5 highlights the TALEN approachlinking Exon 22 of the F8 gene to a nucleic acid encoding a truncatedFVIII polypeptide encoding exons 23-26. Panel A of FIG. 5 shows thespecific F8 genomic DNA sequence (spanning genic base positions126,625-126,693) within which a double-stranded DNA break is introduced(designated “Endonuclease domain” in Panel B) by this strategy'sfunctional TALEN dimer. The left and right TALEN protein sequences forthe variable DNA-binding domain are listed as Seq. ID. No. 4 and Seq.ID. No. 6, respectively. An example of DNA sequences encoding the leftand right TALEN DNA-binding domains are listed as Seq. ID. No 5 and Seq.ID. No. 7, respectively. Because of the degeneracy of the genetic code,there are many possible constructs that can be used to encode TALENDNA-binding domains. In some embodiments, the codons are optimized forexpression of the DNA constructs. Panel A in FIG. 5 also shows the F8genomic DNA sequence containing (i) the recognition sites for the left(TALEN_(L)-hF8_(E22/I22)) and right (TALEN_(R)-hF8_(E22/I22)) TALENmonomers comprising F8-TALEN-5 and (ii) the intervening spacer regionwithin which the F8-TALEN-5's endonuclease activity creates thedouble-stranded DNA breaks (DSDBs) required for inducing the physiologiccellular machinery that mediates the homology-dependent DNA repairpathway. Panel A in FIG. 5 also shows important orienting landmarks,including the following: (i) Nucleotide coordinates of this region(based on the February, 2009, human genome assembly [UCSC GenomeBrowser: http://genome.ucsc.edu/]) are numbered with respect to thewild-type F8 transcription unit, where the initial (5′-most) base of theF8 pre-mRNA (5′-base of exon-1 [E1]) is designated +1 or 1 (note thatthis base corresponds to X-chromosome position 154,250,998) and includesthe appropriate intronic sequence bases in calculating the genomic basepositioning; (ii) Relative location of the X-chromosome's centromere(X-Cen) and its long-arm telomere (Xq-Tel), as transcription of thewild-type F8 locus and all of its mutant alleles causing HA—with theexception of its two recurrent intronic inversions, the intron-1(I1)-inversion (I1I)- and the I22I-mutations—is oriented towards X-Cen.Transcription of the I1- and I22-inverted F8 loci, in contrast, areoriented towards Xq-Tel. This strategy repairs (i) the highly recurrentI22I-mutation—also designated F8_(I22I)—which causes ˜45% of allunrelated patients with severe hemophilia-A (HA) and (ii) mutant F8 lociin ˜20% of all other patients with severe HA, who are either known orfound to have any one of the >200 distinct mutations that have beenfound (according to the HAMSTeRS database of HA-causing F8 mutations)thus far to reside down-stream (i.e., 3′) of exon-22 (E22). The lastcodon of exon 22 encodes methionine (Met [M]) as translated residue2,143 (2,124 in the mature FVIII protein secreted into plasma). Mostmutations repaired are “previously known” (literature and/or HAMSTeRS orother databases), some have never been identified previously; the F8abnormalities in this latter category are “private” (found only in thisparticular) to the patient/family.

Panel B in FIG. 5 shows the functional aspects of the TALENs includingthe overall DNA-binding domain (DBD) and the DBD-subunit repeats of theleft and right monomers (TALEN_(L)-hF8_(E22/I22) andTALEN_(R)-hF8_(E22/I22)). Also shown are the (i) specific DNA sequencesrecognized by each TALEN monomer (shown in bold font immediately beloweach DBD-subunit); (ii) the spacer region between the DNA recognitionsequences of the TALEN monomers contains the sequence within which thedimerized Fok1 catalytic domains, which form a functional endonuclease,introduce a double-stranded DNA break (DSDB). As shown in the lower leftportion of FIG. 5, the introduction of a DSDB in the presence ofhomologous repair vehicle no. 5 (HRV5), the nucleotide sequence of whichis provided below as Seq. ID. No. 12, results in the in-frameintegration, immediately 3′ to exon 22, of the partial human F8 cDNAcomprising exons 23, 24 and 25 and the protein coding sequence, or CDS,of exon 26 (designated hF8[E23-E25/E26_(CDS)]). In one embodiment, theTALEN constructs depicted in FIG. 5 can be used to repair all I22Iinversion mutations (See #1 pathway). In another embodiment, the sameconstructs can be used to repair non-I22I F8 mutations that occur 3′(i.e. downstream) of the exon-22/intron-22 junction (See #2 pathway).

FIG. 6 illustrates a TALEN-mediated strategy to repair the human FactorVIII (FVIII) gene (F8) mutations in >50% of all patients with severehemophilia-A (HA), including the highly recurrent intron-22(I22)-inversion (I22I)-mutation. FIG. 6 highlights the TALEN approachlinking intron 22 of the F8 gene to a nucleic acid encoding a truncatedFVIII polypeptide encoding exons 23-26. Panel A shows the specific F8genomic DNA sequence within which a double-stranded DNA break isintroduced (designated “Endonuclease domain” in Panel B) by thisstrategy's functional TALEN dimer. The left and right TALEN proteinsequences for the variable DNA-binding domain are listed as Seq. ID. No.8 and Seq. ID. No. 10, respectively. Examples of DNA sequences encodingthe left and right TALEN DNA-binding domains are listed as Seq. ID. No.9 and Seq. ID. No. 11, respectively. Because of the degeneracy of thegenetic code, there are many possible constructs that can be used toencode TALEN DNA-binding domains. In some embodiments, the codons areoptimized for expression of the DNA constructs. Panel A in FIG. 6 alsoshows important orienting landmarks, including the: (i) nucleotidecoordinates of this region (based on the February, 2009, human genomeassembly available at the UCSC Genome Browser: http://genome.ucsc.edu/)are numbered with respect to the wild-type F8 transcription unit, wherethe initial (5′-most) base of the F8 pre-mRNA (5′ most base of exon-1[E1]) is designated +1 or 1 (note that this base corresponds toX-chromosome position 154,250,998) and includes the appropriate intronicsequence bases in calculating the genomic base positioning; (ii)relative location of the X-chromosome's centromere (X-Cen) and itslong-arm telomere (Xq-Tel), as transcription of the wild-type F8 locusand all of its mutant alleles causing HA—with the exception of its tworecurrent intronic inversions, the intron-1 (I1)-inversion (I1I)- andthe I22I-mutations—is oriented towards X-Cen; Transcription of the I1-and I22-inverted F8 loci, in contrast, is oriented towards Xq-Tel. Thisstrategy repairs (i) the highly recurrent I22I-mutation—also designatedF8_(I22I)—which causes ˜45% of all unrelated patients with severehemophilia-A (HA) and (ii) mutant F8 loci in ˜20% of all other patientswith severe HA, who are either known or found to have any one ofthe >200 distinct mutations that have been found (according to theHAMSTeRS database of HA-causing F8 mutations) thus far to residedown-stream (i.e., 3′) of exon-22 (E22). The last codon of E22 entirelyencodes methionine (Met [M]) as translated residue 2,143 (2,124 in themature FVIII protein secreted into plasma). Most mutations repaired are“previously known” (literature and/or HAMSTeRS or other databases), butsome have never been identified previously. The F8 abnormalities in thislatter category are “private” (found only in this particular) to thepatient/family.

Panel B in FIG. 6 shows the functional aspects of the TALENs includingthe overall DNA-binding domain (DBD) and the DBD-subunit repeats of theleft and right monomers (TALEN_(L)-hF8_(I22) and TALEN_(R)-hF8_(I22)).Also shown are the (i) specific DNA sequences recognized by each TALENmonomer (shown in bold font immediately below each DBD-subunit); (ii)the spacer region between the DNA recognition sequences of the TALENmonomers contains the sequence within which the dimerized Fok1 catalyticdomains, which form a functional endonuclease, introduce adouble-stranded DNA break (DSDB). As shown in the lower left portion ofFIG. 6, the introduction of a DSDB in the presence of a homologousrepair vehicle, the nucleotide sequence of which is listed as Seq. ID.No. 13, results in the integration into intron 22 of a native F8 3′splice acceptor site operably linked to a nucleic acid encoding F8 exons23, 24 and 25 and the protein coding sequence, or CDS, of exon 26(designated hF8[E23-E25/E26_(CDS)]). In one embodiment, the TALENconstructs depicted in FIG. 6 can be used to repair all I22I inversionmutations (See #1 pathway). In another embodiment, the same constructsare used to repair non-I22I F8 mutations that occur 3′ (i.e. downstream)of the exon-22/intron-22 junction (See #2 pathway).

In some embodiments, nucleic acids encoding nucleases specificallytarget intron1, intron14, or intron22. In some embodiments, nucleicacids encoding nucleases specifically target the exon1/intron1 junction;exon14/intron14 junction; or the exon22/intron22 junction.

FIG. 9 illustrates an example of a donor plasmid that can be used torepair the F8 gene at the exon22/intron22 junction using a TALENnuclease, zinc finger nuclease, or cas nuclease approach. The donorplasmid contains the cDNA sequence for exons 23-26 of the F8 gene and apolyadenylation signal sequence flanked by two regions of homology tothe F8 gene. The left homology region contains a DNA sequence(approximately 700 base pairs) that is homologous to part of Intron 21and Exon22 of the F8 gene. The right homology region contains a DNAsequence (approximately 700 base pairs) that is homologous to part ofintron 22 of the F8 gene. Upon successful homologous recombination intothe F8 locus, the integrated construct expresses the resulting mRNAencoding the wild-type (corrected) version of the F8 protein. Thesequence of the plasmid depicted in FIG. 9 is listed as Seq. ID. No. 12.The annotation of Seq. ID. No. 12 is provided in Table 1 below.

TABLE 1 Repair vehicle targeted to the Exon 22-Intron 22 junction of F8LOCUS RepairVehicle 7753 bp DNA linear FEATURES Location/Qualifiersmisc_feature 21..327 /note=“f1 origin (-) ” misc_feature 6765..7625/note=“<= Ampicillin” misc_feature 471..614 /label=<= lacZ Amisc_feature 626..644 /note=“T7 promoter =>” misc_feature 5564..5583/note=“T3 promoter =>” misc_feature 6765..7625 /note=“<= Orf1”misc_feature 7667..7695 /note=“<= AmpR promoter” misc_feature 658..740/note=“MCS” misc_feature 1446..2072 /note=“Exons 23-26 (cDNA seq) ”misc_feature 1730..1737 /note=“Create NotI site” misc_feature 2082..2707/note=“hGH polyA” misc_feature 1785..1787 /note=“ns-SNP: A6940G (M2238V)” misc_feature 3408..4160 /note=“HSV-TK promoter ” misc_feature4161..5546 /note=“HSV-TK gene and TK pA Terminator ” misc_feature741..745 /note=“Create site for cloning” misc_feature 5547..5551/note=“Create site for cloning” misc_feature 746..1445 /note=“Lefthomolgy arm (700 bp) ” misc_feature 1290..1445 /note=“Exon 22”misc_feature 1433..1445 /note=“Partial Left TALEN recognition site”misc_feature 2708..3407 /note=“Right homology arm (700 bp) ”misc_feature 2708..2716 /note=“Partial Right TALEN recognition site”misc_feature 2708..3407 /note=“Partial Intron 22” misc_feature 746..1289/note=“Partial Intron 21” source 1..7753 /dnas_title=“RepairVehicleE22-I22 pBluescript”

FIG. 10 illustrates an example of a donor plasmid that may be used torepair the F8 gene using a TALEN nuclease, zinc finger nuclease, or casnuclease approach. The donor plasmid contains the cDNA sequence forexons 2-26 of the F8 gene flanked by two regions of homology to the F8gene. The left homology region contains a DNA sequence that ishomologous to part of the F8 promoter and part of Exon 1. The righthomology region contains a DNA sequence that is homologous to part ofintron 1. Upon successful homologous recombination into the F8 gene, theintegrated construct expresses the resulting mRNA encoding the wild-type(corrected) version of the F8 protein. The donor sequence is cloned intoplasmid (p)BlueScript-II KS-minus (pBS-II-KS[−]). The donor plasmid isused with a TALEN, ZFN, or CRISPR/Cas genomic editing strategy. Thesequence of the plasmid depicted in FIG. 10 is listed as Seq. ID. No.13. The annotation of Seq. ID. No. 13 is provided in Table 2 below.

TABLE 2 Repair vehicle targeted to the Exon 1-Intron 1 junction of F8LOCUS RepairVehicle 11418 bp DNA linear FEATURES Location/Qualifiersmisc_feature 21..327 /note=“f1 origin (-) ” misc_feature 10430..11290/note=“<= Ampicillin” misc_feature 471..614 /label=<= lacZ Amisc_feature 626..644 /note=“T7 promoter =>” misc_feature 9229..9248/note=“<= T3 promoter” misc_feature 10430..11290 /note=“<= Orf1”misc_feature 11332..11360 /note=“<= AmpR promoter” misc_feature 658..740/note=“MCS” misc_feature 5780..6405 /note=“hGH polyA” misc_feature7073..7825 /note=“HSV-TK promoter ” misc_feature 7826..9211/note=“HSV-TK gene and TK pA Terminator ” misc_feature 740..745/note=“Create site for cloning” misc_feature 1540..5770 /note=“Exons2-26 BDD (cDNA seq) ” misc_feature 2664..2669 /note=“Create ClaI site”misc_feature 2903..2905 /note=“ns-SNP: G1679A (R484H) ” misc_feature3680..3685 /note=“BDD (Ser743 - Gln1638) ” misc_feature 5428..5435/note=“Create NotI site” misc_feature 5768..5768 /dnas_title=“Stop”/vntifkey=“21” /label=Stop misc_feature 5483..5485 /note=“ns-SNP: A6940G(M2238V) ” insertion_seq 3934..5770 /dnas_title=“Tg” /vntifkey=“14”/label=Tg misc_feature 9212..9217 /note=“Create site for cloning”misc_feature 9212..9212 /note=“MCS” misc_feature 746..1539 /note=“Lefthomolgy arm (794bp) ” misc_feature 746..1237 /note=“Partial F8 promoter”misc_feature 1238..1539 /note=“Partial Exon 1” misc_feature 6406..7072/note=“Right homology arm (667 bp) ” misc_feature 6406..7072/note=“Partial intron 1” source 1..11418 /dnas_title=“RepairVehicleE1-I1 pBluescript”

FIG. 11 illustrates an example of a donor plasmid that is used to repairthe F8 gene in intron 22 using a TALEN nuclease, zinc finger nuclease,or cas nuclease approach. The donor plasmid contains a 3′ splice site,the cDNA sequence for exons 23-26 of the F8 gene, and a polyadenylationsignal sequence flanked by two regions of homology to the F8 gene. Theleft homology region contains a DNA sequence (approximately 700 basepairs) that is homologous to part of Intron 22 of the F8 gene. The righthomology region contains a DNA sequence (approximately 700 base pairs)that is homologous to part of intron 22 of the F8 gene. Upon successfulhomologous recombination into the F8 locus, the integrated constructexpresses the resulting mRNA encoding the wild-type (corrected) versionof the F8 protein. The sequence of the plasmid depicted in FIG. 11 islisted as Seq. ID. No. 14. The annotation of Seq. ID. No. 14 is providedin Table 3 below.

TABLE 3 Repair vehicle targeted to Intron 22 of F8 LOCUS RepairVehicle7755 bp DNA linear FEATURES Location/Qualifiers misc_feature 21..327/note=“f1 origin (-) ” misc_feature 6767..7627 /note=“<=Ampicillin”misc_feature 471..614 /label=<= lacZ A misc_feature 626..644 /note=“T7promoter =>” misc_feature 5566..5585 /note=“T3 promoter =>” misc_feature6767..7627 /note=“<= Orf1” misc_feature 7669..7697 /note=“<= AmpRpromoter” misc_feature 658..740 /note=“MCS” misc_feature 1448..2074/note=“Exons 23-26 (cDNA seq) ” misc_feature 1732..1739 /note=“CreateNotI site” misc_feature 2084..2709 /note=“hGH polyA” misc_feature1787..1789 /note=“ns-SNP: A6940G (M2238V) ” misc_feature 3410..4162/note=“HSV-TK promoter ” misc_feature 4163..5548 /note=“HSV-TK gene andTK pA Terminator ” misc_feature 741..745 /note=“Create site for cloning”misc_feature 5549..5553 /note=“Create site for cloning” misc_feature746..1445 /note=“Left homology arm (700 bp) ” misc_feature 1437..1445/note=“Partial Left TALEN recognition site” misc_feature 2710..3409/note=“Right homolgy arm (700 bp) ” misc_feature 2710..2719/note=“Partial Right TALEN recognition site” misc_feature 746..1445/note=“Partial Intron 22” misc_feature 2710..3409 /note=“Partial Intron22” misc_feature 1446..1447 /note=“3′ spice site” source 1..7755/dnas_title=“RepairVehicle 122 pBluescript”

FIG. 12 illustrates an example of a donor plasmid that is used to repairthe F8 gene in intron1 using a TALEN nuclease, zinc finger nuclease, orcas nuclease approach. The donor plasmid contains a 3′ splice site, thecDNA sequence of the F8 gene for exons 2-26 lacking the B-domain(B-domain deleted (BDD) version of the F8 gene), and a polyadenylationsignal sequence flanked by two regions of homology to the F8 gene. Theleft homology region contains a DNA sequence (approximately 700 basepairs) that is homologous to part of Exon 1 and Intron 1 of the F8 gene.The right homology region contains a DNA sequence (approximately 700base pairs) that is homologous to part of intron 1 of the F8 gene. Uponsuccessful homologous recombination into the F8 locus, the integratedconstruct expresses the resulting mRNA encoding the wild-type(corrected) version of the F8 protein. The sequence of the plasmiddepicted in FIG. 12 is listed as Seq. ID. No. 15. The annotation of Seq.ID. No. 15 is provided in Table 4 below.

TABLE 4 Repair vehicle targeted to Intron 1 of F8 LOCUS RepairVehicle11359 bp DNA linear FEATURES Location/Qualifiers misc_feature 21..327/note=“f1 origin (-) ” misc_feature 10371..11231 /note=“<= Ampicillin”misc_feature 471..614 /label=<= lacZ A misc_feature 626..644 /note=“T7promoter =>” misc_feature 9170..9189 /note=“<= T3 promoter” misc_feature10371..11231 /note=“<= Orf1” misc_feature 11273..11301 /note=“<= AmpRpromoter” misc_feature 658..740 /note=“MCS” misc_feature 874..1187/note=“Exon 1” misc_feature 1436..1445 /note=“Partial Left TALENrecognition site” misc_feature 5688..6313 /note=“hGH polyA” misc_feature6314..7013 /note=“Right homology arm (700 bp) ” misc_feature 6314..6322/note=“Partial Right TALEN recognition site” misc_feature 7014..7766/note=“HSV-TK promoter ” misc_feature 7767..9152 /note=“HSV-TK gene andTK pA Terminator ” misc_feature 746..1445 /note=“Left homolgy arm (700bp) ” misc_feature 746..873 /note=“Partial F8 promoter” misc_feature740..745 /note=“Create site for cloning” misc_feature 6314..7013/note=“Partial Intron 1” misc_feature 1448..5678 /note=“Exons 2-26 BDD(cDNA seq) ” misc_feature 1446..1447 /note=“3′ spice site” misc_feature2572..2577 /note=“Create ClaI site” misc_feature 2811..2813/note=“ns-SNP: G1679A (R484H) ” misc_feature 3588..3593 /note=“BDD(Ser743 - Gln1638) ” misc_feature 5336..5343 /note=“Create NotI site”misc_feature 5676..5676 /dnas_title=“Stop” /vntifkey=“21” /label=Stopmisc_feature 5391..5393 /note=“ns-SNP: A6940G (M2238V) ” insertion_seq3842..5678 /dnas_title=“Tg” /vntifkey=“14” /label=Tg misc_feature9153..9158 /note=“Create site for cloning” misc_feature 9153..9153/note=“MCS” source 1..11359 /dnas_title=“RepairVehicle I1 pBluescript”

In one embodiment, the integration matrix component for each of thesedistinct homologous donor plasmid is either a cDNA that is linked to theimmediately upstream exon or a cDNA that has a functional3′-intron-splice-junction so that the cDNA sequence is linked throughthe RNA intermediate following removal of the intron. In one embodiment,the donor plasmid is personalized, on an individual basis, so that eachpatient's gene that is repaired expresses the form of the FVIII proteinthat they are maximally tolerant of.

Zinc Finger Nucleases (ZFNs)

Engineered nucleases have emerged as powerful tools for site specificediting of the genome. For example, zinc finger nucleases (ZFNs) arehybrid proteins containing the zinc-finger DNA-binding domain present intranscription factors and the non-specific cleavage domain of theendonuclease Fok1. (Li et al., In vivo genome editing restoreshaemostasis in a mouse model of haemophilia, Nature 2011 Jun. 26;475(7355):217-21).

The same sequences targeted by the TALEN approach, discussed above, canalso be targeted by the zinc finger nuclease approach for genomeediting. Zinc finger nucleases (ZFNs) are a class of engineeredDNA-binding proteins that facilitate targeted editing of the genome bycreating double-strand breaks in DNA at user-specified locations. EachZinc Finger Nuclease (ZFN) consists of two functional domains: 1) aDNA-binding domain comprised of a chain of two-finger modules, eachrecognizing a unique hexamer (6 bp) sequence of DNA, wherein two-fingermodules are stitched together to form a Zinc Finger Protein, each withspecificity of ≥24 bp, and 2) a DNA-cleaving domain comprised of thenuclease domain of Fok I. The DNA-binding and DNA-cleaving domains arefused together and recognize the targeted genomic sequences, allowingthe Fok1 domains to form a heterodimeric enzyme that cleaves the DNA bycreating double stranded breaks.

Zinc finger nucleases can be readily made by using techniques known inthe art (Wright D A, et al. Standardized reagents and protocols forengineering zinc finger nucleases by modular assembly. Nat Protoc. 2006;1(3):1637-52). Engineered zinc finger nucleases can stimulate genetargeting at specific genomic loci in animal and human cells. Theconstruction of artificial zinc finger arrays using modular assembly hasbeen described. The archive of plasmids encoding more than 140well-characterized zinc finger modules together with complementaryweb-based software for identifying potential zinc finger target sites ina gene of interest has also been described. These reagents enable easymixing-and-matching of modules and transfer of assembled arrays toexpression vectors without the need for specialized knowledge of zincfinger sequences or complicated oligonucleotide design (Wright D A, etal. Standardized reagents and protocols for engineering zinc fingernucleases by modular assembly. Nat Protoc. 2006; 1(3):1637-52). Any genein any organism can be targeted with a properly designed pair of ZFNs.Zinc-finger recognition depends only on a match to the target DNAsequence (Carroll, D. Genome engineering with zinc-finger nucleases.Genetics Society of America, 2011, 188(4), pp 773-782).

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) andCRISPR Associated (Cas) Nucleases

In addition to the current protein-based targeting methods (TALEN andZinc Finger) useful for gene targeting, there is another system forgenome editing that uses a short RNA to guide a nuclease to the DNAtarget. This system is called the CRISPR technology. (Mali P, Yang L,Esvelt K M, Aach J, Guell M, DiCarlo J E, Norville J E, Church G M.RNA-guided human genome engineering via Cas9. Science. 2013 Feb. 15;339(6121):823-6; Gasiunas G, Barrangou R, Horvath P, Siksnys V.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage foradaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012 Sep. 25;109(39):E2579-86. Epub 2012 Sep. 4).

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)and CRISPR Associated (Cas) system was discovered in bacteria andfunctions as a defense against foreign DNA, either viral or plasmid. Inbacteria, the endogenous CRISPR/Cas system targets foreign DNA with ashort, complementary single-stranded RNA (CRISPR RNA or crRNA) thatlocalizes the Cas9 nuclease to the target DNA sequence. The DNA targetsequence can be on a plasmid or integrated into the bacterial genome.The crRNA can bind on either strand of DNA and the Cas9 cleaves bothstrands (double strand break, DSB). A recent in vitro reconstitution ofthe Streptococcus pyogenes type II CRISPR system demonstrated that crRNAfused to a normally trans-encoded tracrRNA is sufficient to direct Cas9protein to sequence-specifically cleave target DNA sequences matchingthe crRNA. The fully defined nature of this two-component system allowsit to function in the cells of eukaryotic organisms such as yeast,plants, and even mammals. By cleaving genomic sequences targeted by RNAsequences, such a system greatly enhances the ease of genomeengineering.

The crRNA targeting sequences are transcribed from DNA sequences knownas protospacers. Protospacers are clustered in the bacterial genome in agroup called a CRISPR array. The protospacers are short sequences (˜20bp) of known foreign DNA separated by a short palindromic repeat andkept like a record against future encounters. To create the CRISPRtargeting RNA (crRNA), the array is transcribed and the RNA is processedto separate the individual recognition sequences between the repeats. Inthe Type II system, the processing of the CRISPR array transcript(pre-crRNA) into individual crRNAs is dependent on the presence of atrans-activating crRNA (tracrRNA) that has sequence complementary to thepalindromic repeat. When the tracrRNA hybridizes to the shortpalindromic repeat, it triggers processing by the bacterialdouble-stranded RNA-specific ribonuclease, RNase III. Any crRNA and thetracrRNA can then both bind to the Cas9 nuclease, which then becomesactivated and specific to the DNA sequence complimentary to the crRNA.(Mali P, Yang L, Esvelt K M, Aach J, Guell M, DiCarlo J E, Norville J E,Church G M. RNA-guided human genome engineering via Cas9. Science. 2013Feb. 15; 339(6121):823-6; Gasiunas G, Barrangou R, Horvath P, Siksnys V.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage foradaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012 Sep. 25;109(39):E2579-86. Epub 2012 Sep. 4).

The double-strand DNA breakages (DSDB) induced by the TALEN approachoverlaps with the 6 distinct sites of DSDB induced by Cas9, viatargeting by 6 distinct CRISPR-guide RNAs [F8-CRISPR/Cas9-1(F8-Ex1/Int1), F8-CRISPR/Cas9-2 (F8-Int1), F8-CRISPR/Cas9-3(F8-Ex14/Int14), F8-CRISPR/Cas9-4 (F8-Int14), F8-CRISPR/Cas9-5(F8-Ex22/Int22), F8-CRISPR/Cas9-6 (F8-Int22)]. This allows use of thesame 6 distinct homologous donor sequences with all three genome editingapproaches, including the TALEN nuclease, the zinc finger nuclease(ZFN), and the CRISPR-associated (Cas) nuclease.

FIG. 13 illustrates a CRISPR/Cas9-mediated strategy to repair the humanFactor VIII (FVIII) gene (F8) mutations in ˜95% of all patients withsevere hemophilia-A (HA), including the highly recurrent intron-1(I1)-inversion (I1I)-mutation as well as the intron-22 (I22)-inversion(I22I)-mutation. FIG. 13 shows the specific F8 genomic DNA sequence(spanning genic base positions 172-354 at intron 1) within which adouble-stranded (ds)-DNA break is introduced (designated “Endonucleasetarget” in this panel) by this strategy's wild-type (wt) CRISPR/Cas9ds-DNase in which both of its endonuclease domains are catalyticallyfunctional (“hF8-CRISPR/Cas9wt-1”). This panel also shows importantorienting landmarks, including the following: (i) Nucleotide coordinatesof this region (based on the February, 2009, human genome assembly [UCSCGenome Browser: http://genome.ucsc.edu/]) are numbered with respect tothe wild-type F8 transcription unit, where the initial (5′-most) base ofthe F8 pre-mRNA (5′-base of exon-1 [E1]) is designated +1 or 1 (notethat this base corresponds to X-chromosome position 154,250,998) andinclude the appropriate intronic sequence bases in calculating thegenomic base positioning; (ii) Relative location of the X-chromosome'scentromere (X-Cen) and its long-arm telomere (Xq-Tel), as transcriptionof the wild-type F8 locus and all of its mutant alleles causing HA—withthe exception of its two recurrent intronic inversions, the I1I- and theI22I-mutations—is oriented towards X-Cen. Transcription of the I1- andI22-inverted F8 loci, in contrast, are oriented towards Xq-Tel. Thisstrategy repairs (i) the highly recurrent I22I-mutation—also designatedF8_(I22I)—which causes ˜45% of all unrelated patients with severehemophilia-A (HA) and (ii) mutant F8 loci in ˜90-95% of all otherpatients with severe HA, who are either known or found to have any oneof the >1,500 distinct mutations that have been found (according to theHAMSTeRS database of HA-causing F8 mutations) thus far to residedown-stream (i.e., 3′) of exon-1 (E1). The last codon of E1 partiallyencodes the translated residue 48 (29 in the mature FVIII proteinsecreted into plasma). Most mutations repaired are “previously known”(literature and/or HAMSTeRS or other databases). Some have never beenidentified previously. These F8 abnormalities in this latter categoryare “private” (found only in this particular) to the patient/family.Finally, FIG. 13 shows the functional aspects of hF8-CRISPR/Cas9wt-1including the overall DNA-binding domain of the CRISPR-associated guide(g)RNA as well as the (i) Protospacer adjacent motif (PAM), which is thesite at which the DNase function of Cas9 introduces the ds-DNA break(DSDB); and (ii) The Transactivating Crispr-RNA (TrCr-RNA), which iscovalently attached the gRNA as is what brings the Cas9 endonuclease tothe genomic DNA target for digestion. The introduction of a DSDB in thepresence of a homologous repair vehicle, results in the in-frameintegration, immediately 3′ to E1, of one of either two partial human F8cDNAs comprising either (i) exons 2-25 and the protein coding sequence,or CDS, of exon 26 (designated hF8[E2-E25/E26_(CDS)]), which effectsrepair of the F8 gene such that it now encodes a full-length wild-typeFVIII protein; or (ii) Exons 2-13 entirely linked next to the very5′-most end of exon-14 (E14), which in turn is linked covalently to thevery 3′-most end of E14 (i.e., a B-domain-deleted [BDD]-F8 cDNA), whichis then covalently linked to Exons 15-25 entirely, and then the proteincoding sequence, or CDS, of exon 26 (designatedhF8[E2-E13/E14-BDD/E15-E25/E26_(CDS)]), which effects repair of the F8gene such that it now encodes a BDD-engineered FVIII protein, which isfully functional in FVIII:C activity. The homologous repair vehicle isselected to have a F8 cDNA with the appropriate alleles at all ns-SNPsites so that the patient can receive a “matched” gene repair or atleast a least mismatched repair.

The left homology arm of the homologous repair vehicle for HomologousRepair Vehicle No. 1 (HRV1) for hF8-CRISP/Cas9wt-1 is listed as Seq. ID.No. 17 and comprises the first 1114 bases of the human F8 genomic DNA(which is shown here as single-stranded and representing the sensestrand) and contains 800 bp of the immediately 5′-promoter region of thehuman F8 gene and all 314 bp of the F8 exon-1 (E1), including its 171 bp5′-UTR and its 143 bp of protein (en)coding sequence (CDS). The actualleft homologous arm (LHA) of the homologous repair vehicle (HRV1), whichis used for this CRISPR/Cas9-mediated F8 gene repair (that occurs at theE1/intron-1 [I1] junction of a given patient's endogenous mutant F8),contains at least 500 bp of this genomic DNA sequence (i.e., from it'svery 3′-end, which corresponds to the second base of the codon fortranslated residue 48 of the wild-type FVIII protein and residue 29 ofthe mature FVIII protein found in the circulation) and could include itall, if, for example, we find that full-length F8 gene repair can beeffected efficiently in the future. In this instance, the integrationmatrix would then follow the LHA of this HRV1, and be covalentlyattached to it, and this integration matrix contains (in-frame with eachother and with the 3′-end of the patient's native exon-1, which isutilized in situ, along with his native F8 promoter, to regulateexpression of the repaired F8 gene), all of F8 exons 2-25, and theprotein CDS of exon-25, followed by the functional mRNA 3′-end formingsignals of the human growth hormone gene (hGH-pA). The F8 cDNA fromexons 2-25 and the CDS of exon-26 to be used in the homologous repairvehicle is listed as Seq. ID. No. 18 and follows the left homology arm,and in this example represents the haplotype (H)3 encoding wild-typevariant of F8, which can be used to cure, for example, patients with theI1I-mutation and the I22I-mutation, that arose on an H3-backgroundhaplotype. This following protein encoding cDNA sequence contains 6,909bp of the entire 7,053 bp of F8 protein encoding sequence (i.e., thefirst 144 bp of protein CDS from FVIII, from its initiator methionine,is not shown, as this is contained in exon-1, which is provided by thepatient's own endogenous exon-1, providing it is not mutant and thusprecluding the repair event). The right homology arm of the homologousrepair vehicle for the cas nuclease approach is listed as Seq. ID. No.19 and includes 1109 bases of human F8 genomic DNA (which is shown hereas single-stranded and representing the sense strand) from the F8 geneintron 1.

4. Replacement Sequence

As described above, following the introduction of the targeting nucleaseinto the cell, the nuclease, upon dimerization, catalyzes a doublestranded break in the DNA between their binding sites. If a doublestranded break occurs in the presence of, for example, a donor plasmid(DP), which contains a stretch of DNA with a left homology (HL) andright homology (HR) arm that have identical DNA sequences to that in thenative chromosomal DNA 5′ and 3′ of the region flanking the break-point,homologous recombination occurs very efficiently. Accordingly, thepresent invention includes the introduction of a nucleic acid sequencethat serves as a donor sequence during homologous recombination whichincludes a partial F8 gene that replaces, and thus repairs, the mutatedportion of the subject's F8 gene.

The donor sequence nucleic acid comprises (i) a nucleic acid encoding atruncated FVIII polypeptide or (ii) a native F8 3′ splice acceptor siteoperably linked to a nucleic acid encoding a truncated FVIII polypeptidethat contains a non-mutated portion of the FVIII protein. The donorsequence is flanked on each side by regions of nucleic acid which arehomologous to the F8 gene. Each of these homologous regions can includeabout 20, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ormore nucleotides homologous with the subject's F8 gene. In embodiments,each of the homologous regions flanking the donor sequence is betweenabout 200 to about 1,200 nucleotides, between 400 and about 1000nucleotides, or between about 600 and about 900 nucleotides. In oneembodiment, each homologous region is between about 800 and about 900nucleotides. Thus, each donor sequence nucleic acid includes a donorsequence replacing an endogenous mutation in the subject's F8 gene, and5′ and 3′ flanking sequences which are homologous to the F8 gene.

The donor sequence is derived based on the specific mutation within thesubject's F8 gene targeted for replacement and repair. Accordingly, thelength of the donor sequence can vary. For example, when repairing apoint mutation, the donor sequence can include only a small number ofreplacement nucleotide sequences compared with, for example, a donorsequence derived for repairing an inversion such as an intron 22inversion. Therefore, a donor sequence can be of any length, for examplebetween 2 and 10,000 nucleotides in length (or any integer value therebetween or there above), preferably between about 100 and 1,000nucleotides in length (or any integer there between), more preferablybetween about 200 and 500 nucleotides in length. The designing of donorsequence nucleic acids is known in the art, for example, see Cermak etal., Efficient design and assembly of custom TALEN and other TALeffector-based constructs for DNA targeting, Nucleic Acid Res. 2011 Sep.1; 39 (17):7879.

In one embodiment of the present invention, the gene mutation targetedfor repair is a point mutation, and the donor sequence includes anucleic acid sequence that replaces the point mutation with a sequencethat does not include the point mutation, for example, the wild-type F8sequence. In one embodiment, the gene mutation targeted for repair is adeletion and the donor sequence includes a nucleic acid sequence thatreplaces the deletion with a sequence that does not include thedeletion, for example, the wild-type F8 sequence.

In one embodiment, the gene mutation targeted for repair is aninversion, and the donor sequence includes a nucleic acid sequence thatencodes a truncated FVIII polypeptide that, upon insertion into the F8genome, repairs the inversion and provides for the production of afunctional FVIII protein. In one embodiment, the gene mutation targetedfor repair is an inversion of intron 1. In one embodiment, the genemutation targeted for repair is an inversion of intron 22, and the donorsequence includes a nucleic acid that encodes all of exons 23-25 and thecoding sequence of exon-26.

In any of the methods described herein, the donor sequence can containsequences that are homologous, but not identical (for example, containnucleic acid sequence encoding wild-type amino acids or differing ns-SNPamino acids), to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to about 99% sequence identity to thegenomic sequence that is replaced. In other embodiments, the homologybetween the donor and genomic sequence is higher than 99%, for exampleif only 1 nucleotide differs as between donor and genomic sequences ofover 100 contiguous base pairs. In certain cases, a non-homologousportion of the donor sequence can contain sequences not present in theregion of interest, such that new sequences are introduced into theregion of interest. In these instances, the non-homologous sequence isgenerally flanked by sequences of 50-1,000 base pairs, or any number ofbase pairs greater than 1,000, that are homologous or identical tosequences in the region of interest. In other embodiments, the donorsequence is non-homologous to the first sequence, and is inserted intothe genome by non-homologous recombination mechanisms.

5. Targeted Cells

The gene targeting and repair approaches using the different nucleasesdescribed above can be carried out using many different target cells.For example, the transduced cells may include endothelial cells,hepatocytes, or stem cells. In one embodiment, the cells can be targetedin vivo. In one embodiment, the cells can be targeted using ex vivoapproaches and reintroduced into the subject.

BOECs

In one embodiment, the target cells from the subject are endothelialcells. In one embodiment, the endothelial cells are blood outgrowthendothelial cells (BOECs). Characteristics that render BOECs attractivefor gene repair and delivery include the: (i) ability to be expandedfrom progenitor cells isolated from blood, (ii) mature endothelial cell,stable, phenotype and normal senescence (˜65 divisions), (iii) prolificexpansion from a single blood sample to 1019 BOECs, (iv) resilience,which unlike other endothelial cells, permits cryopreservation and hencemultiple doses for a single patient prepared from a single isolation.Methods of isolation of BOECs are known, where the culture of peripheralblood provides a rich supply of autologous, highly proliferativeendothelial cells, also referred to as blood outgrowth endothelial cells(BOECs). Bodempudi V, et al., Blood outgrowth endothelial cell-basedsystemic delivery of antiangiogenic gene therapy for solid tumors.Cancer Gene Ther. 2010 December; 17(12):855-63.

Studies in animal models have revealed properties of blood outgrowthendothelial cells that indicate that they are suitable for use in exvivo gene repair strategies. For example, a key finding concerning thebehavior of canine blood outgrowth endothelial cells (cBOECs) is thatcBOECs persist and expand within the canine liver after infusion.Milbauer L C, et al. Blood outgrowth endothelial cell migration andtrapping in vivo: a window into gene therapy. 2009 April; 153(4):179-89.Whole blood clotting time (WBCT) in the HA model was also improved afteradministration of engineered cBOECs. WBCT dropped from a pretreatmentvalue of under 60 min to below 40 min and sometimes below 30 min.Milbauer L C, et al., Blood outgrowth endothelial cell migration andtrapping in vivo: a window into gene therapy. 2009 April; 153(4):179-89.

LSECs

In one embodiment, the target cells from the subject are hepatocytes. Inone embodiment, the cell is a liver sinusoidal endothelial cell (LSECs).Liver sinusoidal endothelial cells (LSEC) are specialized endothelialcells that play important roles in liver physiology and disease.Hepatocytes and liver sinusoidal endothelial cells (LSECs) are thoughtto contribute a substantial component of FVIII in circulation, with avariety of extra-hepatic endothelial cells supplementing the supply ofFVIII.

In one embodiment, the present invention targets LSEC cells, as LSECcells likely represent the main cell source of FVIII. Shahani, T, etal., Activation of human endothelial cells from specific vascular bedsinduces the release of a FVIII storage pool. Blood 2010;115(23):4902-4909. In addition, LSECs are believed to play a role ininduction of immune tolerance. Onoe, T, et al., Liver sinusoidalendothelial cells tolerize T cells across MHC barriers in mice. JImmunol 2005; 175(1):139-146. Methods of isolation of LSECs are known inthe art. Karrar, A, et al., Human liver sinusoidal endothelial cellsinduce apoptosis in activated T cells: a role in tolerance induction.Gut. 2007 February; 56(2): 243-252.

iPSCs

In one embodiment, the transduced cells from the subject are stem cells.In one embodiment, the stem cells are induced pluripotent stem cells(iPSCs). Induced pluripotent stem cells (iPSCs) are a type ofpluripotent stem cell artificially derived from a non-pluripotent cell,typically an adult somatic cell, by inducing expression of specificgenes and factors important for maintaining the defining properties ofembryonic stem cells. Induced pluripotent stem cells (iPSCs) have beenshown in several examples to be capable of site specific gene targetingby nucleases. Ru, R. et al. Targeted genome engineering in human inducedpluripotent stem cells by penetrating TALENs. Cell Regeneration. 2013,2:5; Sun N, Zhao H. Seamless correction of the sickle cell diseasemutation of the HBB gene in human induced pluripotent stem cells usingTALENs. Biotechnol Bioeng. 2013 Aug. 8. Induced pluripotent stem cells(iPSCs) can be isolated using methods known in the art. Lorenzo, I M.Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) fromPrimary Somatic Cells. Stem Cell Rev. 2013 August; 9(4):435-50.

Co-Cultured Cells

As discussed above, a number of different cells types may be targetedfor repair. However, pure populations of some cell types may not promotesufficient homing and implantation upon reintroduction to provideextended and sufficient expression of the corrected F8 gene. Therefore,some cell types may be co-cultured with different cell types to helppromote cell properties (i.e. ability of cells to engraft in the liver).

In one embodiment, the transduced cells are from blood outgrowthendothelial cells (BOECs) that have been co-cultured with additionalcell types. In one embodiment, the transduced cells are from bloodoutgrowth endothelial cells (BOECs) that have been co-cultured withhepatocytes or liver sinusoidal endothelial cell (LESCs) or both. In oneembodiment, the transduced cells are from blood outgrowth endothelialcells (BOECs) that have been co-cultured with induced pluripotent stemcells (iPSCs).

6. Cell Delivery

Methods of nucleic acid delivery are well known in the art. (See, e.g.,WO 2012051343). In the methods provided herein, the described nucleaseencoding nucleic acids can be introduced into the cell as DNA or RNA,single-stranded or double-stranded and can be introduced into a cell inlinear or circular form. In one embodiment, the nucleic acids encodingthe nuclease are introduced into the cell as mRNA. The donor sequencecan introduced into the cell as DNA single-stranded or double-strandedand can be introduced into a cell in linear or circular form. Ifintroduced in linear form, the ends of the nucleic acids can beprotected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues.

The nucleic acids can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,the nucleic acids can be introduced as naked nucleic acid, as nucleicacid complexed with an agent such as a liposome or poloxamer, or can bedelivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus,lentivirus).

The nucleic acids may be delivered in vivo or ex vivo by any suitablemeans. Methods of delivering nucleic acids are described, for example,in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882;6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824.

Any vector systems may be used including, but not limited to, plasmidvectors, retroviral vectors, lentiviral vectors, adenovirus vectors,poxvirus vectors; herpesvirus vectors and adeno-associated virusvectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824. Furthermore, any ofthese vectors may comprise one or more of the sequences needed fortreatment. Thus, when one or more nucleic acids are introduced into thecell, the nucleases and/or donor sequence nucleic acids may be carriedon the same vector or on different vectors. When multiple vectors areused, each vector can comprise a sequence encoding a TALEN-L monomer, aTALEN-R monomer, or a donor sequence nucleic acid. Alternatively, two ormore of the nucleic acids can be contained on a single vector.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding the nucleic acids in cells (e.g.,mammalian cells) and target tissues. Non-viral vector delivery systemsinclude DNA plasmids, naked nucleic acid, and nucleic acid complexedwith a delivery vehicle such as a liposome or poloxamer. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. Methods of non-viraldelivery of nucleic acids include electroporation, lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., theSonitron 2000 system (Rich-Mar) can also be used for delivery of nucleicacids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially {e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Feigner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal, Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem.5:382-389 (1994); Remy et al, Bioconjugate Chem. 5:647-654 (1994); Gaoet al, Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids include, but are not limited to,retroviral, lentivirus, adenoviral, adeno-associated, vaccinia andherpes simplex virus vectors for gene transfer.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cz's-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cz's-acting LTRs are sufficient for replicationand packaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al, J. Virol. 66:2731-2739(1992); Johann et al, J. Virol. 66:1635-1640 (1992); Sommerfelt et al.,Virol. 176:58-59 (1990); Wilson et al, J. Virol. 63:2374-2378 (1989);Miller et al, J. Virol. 65:2220-2224 (1991); PCT US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al, Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors is described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al, MolCell. Biol. 5:3251-3260 (1985); Tratschin, et al, Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al, J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent. pLASN and MFG-S are examples ofretroviral vectors that have been used in clinical trials (Dunbar et al,Blood 85:3048-305 (1995); Kohn et al, Nat. Med. 1:1017-102 (1995);Malech et al, PNAS 94:22 12133-12138 (1997)). PA317/pLASN was the firsttherapeutic vector used in a gene therapy trial. (Blaese et al, Science270:475-480 (1995)). Transduction efficiencies of 50% or greater havebeen observed for MFG-S packaged vectors. (Ellem et al, ImmunolImmunother. 44(1):10-20 (1997); Dranoff et al, Hum. Gene Ther. 1:111-2(1997). Recombinant adeno-associated virus vectors (rAAV) are analternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al, Lancet 351: 9117 1702-3 (1998), Kearns et al, Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh.lO and any novel AAV serotype canalso be used in accordance with the present invention. In a particularembodiment, the vector is based on a hepatotropic adeno-associated virusvector, serotype 8 (see, e.g., Nathwani et al., Adeno-associated viralvector mediated gene transfer for hemophilia B, Blood 118(21):4-5,2011).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad El a, El b, and/or E3 genes; subsequently thereplication defective vector is propagated in human 293 cells thatsupply deleted gene function in trans. Ad vectors can transduce multipletypes of tissues in vivo, including non-dividing, differentiated cellssuch as those found in liver, kidney and muscle. Conventional Ad vectorshave a large carrying capacity. An example of the use of an Ad vector ina clinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al, Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et ah,Infection 24:1 5-10 (1996); Sterman et ah, Hum. Gene Ther. 9:7 1083-1089(1998); Welsh et ah, Hum. Gene Ther. 2:205-18 (1995); Alvarez et al,Hum. Gene Ther. 5:597-613 (1997); Topf et al, Gene Ther. 5:507-513(1998); Sterman et al, Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many applications, it is desirable that the g vector be deliveredwith a high degree of specificity to a particular tissue type.Accordingly, a viral vector can be modified to have specificity for agiven cell type by expressing a ligand as a fusion protein with a viralcoat protein on the outer surface of the virus. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et ah, Proc. Natl. Acad. Sci. USA92:9747-9751 (1995), reported that Moloney murine leukemia virus can bemodified to express human heregulin fused to gp70, and the recombinantvirus infects certain human breast cancer cells expressing humanepidermal growth factor receptor. This can be used with othervirus-target cell pairs, in which the target cell expresses a receptorand the virus expresses a fusion protein comprising a ligand for thecell-surface receptor. For example, filamentous phage can be engineeredto display antibody fragments (e.g., FAB or Fv) having specific bindingaffinity for virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to non-viral vectors. Such vectors can be engineered tocontain specific uptake sequences which favor uptake by specific targetcells.

Vectors can be delivered in vivo by administration to an individualpatient, typically by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ortopical application, as described below. Alternatively, vectors can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by re-implantation ofthe cells into a patient, usually after selection for cells which haveincorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingthe nucleic acids described herein can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells including, butnot limited to, injection, infusion, topical application andelectroporation. Suitable methods of administering such nucleic acidsare available and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Vectors suitable for introduction of the nucleic acids described hereininclude non-integrating lentivirus vectors (IDLV). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/054985.

The nucleic acids encoding the TALEN-L left monomer and TALEN-R rightmonomer can be expressed either on separate expression constructs orvectors, or can be linked in one open reading frame. Expression of thenuclease can be under the control of a constitutive promoter or aninducible promoter.

Administration can be by any means in which the polynucleotides aredelivered to the desired target cells. For example, both in vivo and exvivo methods are contemplated. In one embodiment, the nucleic acids areintroduced into a subject's cells that have been explanted from thesubject, and reintroduced following F8 gene repair.

For in vivo administration, intravenous injection of the nucleic acidsto the portal vein is a preferred method of administration. Other invivo administration modes include, for example, direct injection intothe lobes of the liver or the biliary duct and intravenous injectiondistal to the liver, including through the hepatic artery, directinjection in to the liver parenchyma, injection via the hepatic artery,and/or retrograde injection through the biliary tree Ex vivo modes ofadministration include transduction in vitro of resected hepatocytes orother cells of the liver, followed by infusion of the transduced,resected hepatocytes back into the portal vasculature, liver parenchymaor biliary tree of the human patient, see e.g., Grossman et ah, (1994)Nature Genetics, 6:335-341.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism as described above, such as, for example,calcium phosphate mediated gene delivery, electroporation,microinjection, proteoliposomes, or viral vector delivery. Thetransduced cells can then be infused (e.g., in a pharmaceuticallyacceptable carrier) or homotopically transplanted back into the subjectper standard methods for the cell or tissue type. Standard methods areknown for transplantation or infusion of various cells into a subject.

7. Immune Tolerance Induction

In one embodiment, the repaired gene, upon expression, provides for theinduction of immune tolerance to an administered replacement FVIIIprotein product. Current FVIII replacement therapies include theinfusions of recombinant FVIII replacement products (r)FVIII. (r)FVIIIis a biosynthetic blood coagulation prepared using recombinant DNA, andis structurally similar to endogenous wild-type human FVIII and producesthe same biological effect. Due to genetic variables within a subjectincluding the individual's specific F8 mutation type, background FVIIIhaplotype, and HLA haplotype, however, the (r)FVIII mismatched aminoacid may induce an immune response in the subject receiving the(r)FVIII, resulting in the development of inhibitors and the reductionin efficiency of the particular (r)FVIII.

Accordingly, in one embodiment, a method of inducing tolerance to a(r)FVIII product in a subject that may receive, is receiving, or hasreceived a (r)FVIII product is provided comprising introducing into acell of the subject one or more isolated nucleic acids encoding anuclease that targets a portion of the human F8 gene containing amutation that causes hemophilia A, wherein the nuclease creates a doublestranded break in the human F8 gene; and an isolated nucleic acidcomprising (i) a nucleic acid encoding a truncated FVIII polypeptide or(ii) a native F8 3′ splice acceptor site operably linked to a nucleicacid encoding a truncated FVIII polypeptide, wherein the nucleic acidcomprising the (i) a nucleic acid encoding a truncated FVIII polypeptideor (ii) native F8 3′ splice acceptor site operably linked to a nucleicacid encoding a truncated FVIII polypeptide is flanked by nucleic acidsequences homologous to the nucleic acid sequences upstream anddownstream of the double stranded break in the DNA, and wherein therepaired gene, upon expression, provides for the induction of immunetolerance to an administered replacement FVIII protein product. Theperson administered the cells in which the F8 gene has been repaired mayor may not have anti-FVIII antibodies as measured by ELISA or Bethesdaassays. In one embodiment, the truncated FVIII polypeptide amino acidsequence shares homology with a positionally coordinated portion of the(r)FVIII amino acid sequence. In one embodiment, the truncated FVIIIpolypeptide amino acid sequence shares complete homology with apositionally correlated portion of the (r)FVIII amino acid sequence. Ina further embodiment, the method further includes targeting andreplacing a nucleic acid sequence within the subject's F8 gene encodinga non-mutational amino acid with the positional equivalent amino acid inthe (r)FVIII. In one embodiment, the non-mutational amino acid is anon-synonymous Single Nucleotide Polymorphism (ns-SNP).

It is believed that intra-cellular expression of even a small amount ofa repaired FVIII protein that closely resembles the (r)FVIII product,and concomitant surface presentation of FVIII epitopes within the cell'sMHC, may enable the induce of immune tolerance to the (r)FVIII products.

The human F8 gene is polymorphic and encodes several structurallydistinct FVIII proteins referred to as haplotypes. Sequencing studies ofthe F8 gene have revealed four common nonsynonymous-single-nucleotidepolymorphisms (nsSNPs) that, together with two infrequent ns-SNPs,encode eight distinct wild-type FVIII proteins referred to as haplotypeH1, H2, H3, H4, H5, H6, H7, and H8. The amino acid sequence of the H1wild-type variant is listed as Seq. ID. No. 16.

All currently available (r)FVIII are based on either the H1 or H2haplotype variant. Commercially available (r)FVIII include the H1variants Kogenate® (Bayer) and Helixate® (ZLB Behring), the H2 variantsRecombinate® (Baxter) and Advate® (Baxter), and the H1/H2 variantB-domain deleted Refacto® (Pfizer) and Xyntha® (Pfizer). The presentinvention, however, is not limited to replacing a subject's F8 gene witha coding sequence that matches or is nearly homologous to a portion ofthe commercially available (r)FVIII products described above, but isapplicable to any (r)FVIII, including human/porcine hybrid (r)FVIII,porcine (r)FVIII, and alternative haplotype recombinant FVIIIreplacement products such as those identified in WO 2006/063031, whichis incorporated by reference herein. Differences between a subject'sFVIII and a (r)FVIII can result from, for example, missense mutations inthe subject's F8 gene, nonsynonymous single-nucleotide polymorphisms(nsSNPs) (both well-known and “private” or individualized) or haplotypicvariations between the subject's FVIII and (r)FVIII, inversions, forexample intron 1 or 22 inversions, synthetic peptide inclusion due toB-domain deletions in the BDD-®FVIII, and the like.

Because the amino acid sequence of available (r)FVIII are known, anddifferences in subject's FVIII can be determined, differences (ormismatches) between the subject's endogenous FVIII protein sequence and(r)FVIII can be readily identifiable using common techniques known inthe art, and the identified differences can be utilized to constructnucleic acids as described above for replacement. For example, moleculargenetic studies have shown that development of inhibitors to factor VIIIreplacement products occurs most frequently in patients with severehemophilia due to major gene lesions including inversions. Subjects withintron 22 inversion express the entire FVIII intracellularly, albeit ontwo separate polypeptides. Importantly, another gene, F8B, is alsogenerally expressed in both normal and HA subjects. The expressionproduct of the F8B gene, FVIIIB, has sequence identity with a portion ofthe C1 domain and the entire C2 domain of FVIII. The presence of thisFVIIIB polypeptide is important from a tolerance standpoint as it servesas a source for any T cells epitope or B cell epitopes needed to supportprocesses that occur in the thymus (T cell clonal deletion) and spleen(B cell anergy) to achieve central tolerance. The expression product ofF8I22I starts at residue 1 and ends at residue 2124. The polypeptideexpressed by the F8B begins at residue 2125 and ends at residue 2332.Accordingly subjects having the F8I22I have the requisite FVIII materialto yield one or more FVIII peptides ending at or before residue 2124,the last amino acid encoded by exon 22, or beginning at or after residue2125, the first amino acid encoded by exon 23. Any T cell epitope withinsuch a peptide can be recognized as a self-antigen and not beimmunogenic in the subject. Peptides spanning the junction betweenresidues 2124 and 2125, if proteolyzed from a (r)FVIII and presented byMHC class II molecules, however, are “foreign” and potentiallyimmunogenic T cell epitopes in an F8I22I subject. Because of this, allsubjects having F8I22I have similar reference.

Likewise, in subjects receiving B-domain deleted-(r)FVIII product,differing amino acid sequence exist between the subject's FVIII and thereplacement product caused by the removal of the B-domain in theBDD-rFVIII product. For example, in certain BDD-rFVIII, a deletion of894 internal codons and splicing codons 762 and 1657 creates a FVIIIproduct containing 1438 amino acids. The BDD-rFVIII contains a syntheticjunctional 14-peptide sequence SFS-QNPPVLKRHQR formed by covalentattachment of the three N-terminal most residues of the B-domain,S⁷⁴¹F⁷⁴²S⁷⁴³, to the 11 C-terminal-most residuesQ¹⁶³⁸N¹⁶³⁹P¹⁶⁴⁰P¹⁶⁴¹V¹⁶⁴²L¹⁶⁴³K¹⁶⁴⁴R¹⁶⁴⁵H¹⁶⁴⁶Q¹⁶⁴⁷R¹⁶⁴⁸. This syntheticlinker creates 11 unique peptides across a 15 amino acid sequence withinthe BDD-rFVIII, which have potential immunogenicity. In one embodiment,a donor sequence including these amino acids is utilized to replace thesubject's F8 gene. Thus, in one embodiment, a donor sequence comprisinga nucleic acid encoding the amino acid sequence SFSQNPPVLKRHQR isprovided. In alternative embodiments, the BDD-rFVIII contains variableamounts of linker sequences. In one embodiment, the linker sequencecomprises the first 10 amino acids from the N-terminus of the B-domainand 11 amino acids from the C-terminus of the B-domain.

In one embodiment, a F8 mutation in a cell from a subject having HA, forexample a BOEC, iPSC, or LSEC, is identified and repaired using thenuclease approach described above and a donor sequence that is identicalto a portion of a (r)FVIII product. In one embodiment, the repair takesplace ex vivo, as a cell, for example a BOEC or LSEC, is explanted fromthe subject, repaired, and reintroduced into the subject as describedabove. Following repair, the cell is capable of producing FVIII,including intracellularly, and express and present potentially antigenicpeptides on MHC complexes. The expression of the repaired FVIII leads toimmune suppression specific to a specific (r)FVIII antigen orimmunogenic epitopes expressed by the repaired F8 gene. Such atolerogenic immune response can include any reduction, delay, orinhibition in an undesired immune response specific to the (r)FVIIIantigen or epitope. Tolerogenic immune responses, therefore, can includethe prevention of or reduction in inhibitors to a specific (r)FVIII.Tolerogenic immune responses as provided herein include immunologicaltolerance. The tolerogenic immune response can be the result of MHCClass II-restricted presentation and/or B cell presentation, or anyother presentation leading to the minimized or reduced immunicity of the(r)FVIII.

Tolerogenic immune responses include a reduction in (r)FVIIIantigen-specific antibody (inhibitor) production. The expression of therepaired FVIII can result in a reduction of measurable Bethesda titerunits to a (r)FVIII in a subject that already has inhibitors to a(r)FVIII. Tolerogenic immune responses also include any response thatleads to the stimulation, production, or recruitment of CD4+ Treg cellsand/or CD8+ Treg cells. CD4+ Treg cells can express the transcriptionfactor FoxP3 and inhibit inflammatory responses and auto-immuneinflammatory diseases (Human regulatory T cells in autoimmune diseases.Cvetanovich G L, Hafler D A. Curr Opin Immunol. 2010 December;22(6):753-60. Regulatory T cells and autoimmunity. Vila J, Isaacs J D,Anderson A E. Curr Opin Hematol. 2009 July; 16(4):274-9). Such cellsalso suppress T-cell help to B-cells and induce tolerance to both selfand foreign antigens (Therapeutic approaches to allergy and autoimmunitybased on FoxP3+ regulatory T-cell activation and expansion. Miyara M,Wing K, Sakaguchi S. J Allergy Clin Immunol. 2009 April; 123(4):749-55).C D4+ Treg cells recognize antigen when presented by Class II proteinson APCs. CD8+ Treg cells, which recognize antigens presented by Class I(and Qa-1), can also suppress T-cell help to B-cells and result inactivation of antigen-specific suppression inducing tolerance to bothself and foreign antigens. Disruption of the interaction of Qa-1 withCD8+ Treg cells has been shown to dysregulate immune responses andresults in the development of auto-antibody formation and an auto-immunelethal systemic-lupus-erythematosus (Kim et al., Nature. 2010 Sep. 16,467 (7313): 328-32). CD8+ Treg cells have also been shown to inhibitmodels of autoimmune inflammatory diseases including rheumatoidarthritis and colitis (CD4+CD25+regulatory T cells in autoimmunearthritis. Oh S, Rankin A L, Caton A J. Immunol. Rev. 2010 January;233(1):97-111. Regulatory T cells in inflammatory bowel disease. Boden EK, Snapper S B. Curr Opin Gastroenterol. 2008 November; 24(6):733-41).In some embodiments, the expression of the repaired FVIII provided caneffectively result in both types of responses (CD4+ Treg and CD8+ Treg).In other embodiments, FoxP3 can be induced in other immune cells, suchas macrophages, iNKT cells, etc., and the compositions provided hereincan result in one or more of these responses as well.

Tolerogenic immune responses also include, but are not limited to, theinduction of regulatory cytokines, such as Treg cytokines; induction ofinhibitory cytokines; the inhibition of inflammatory cytokines (e.g.,IL-4, IL-1b, IL-5, TNF-α, IL-6, GM-CSF, IFN-γ, IL-2, IL-9, IL-12, IL-17,IL-18, IL-21, IL-22, IL-23, M-CSF, C reactive protein, acute phaseprotein, chemokines (e.g., MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC orIP-10), the production of anti-inflammatory cytokines (e.g., IL-4,IL-13, IL-10, etc.), chemokines (e.g., CCL-2, CXCL8), proteases (e.g.,MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2), prostaglandins(e.g., PGE2) or histamines; the inhibition of polarization to a Th17,Th1, or Th2 immune response; the inhibition of effector cell-specificcytokines: Th17 (e.g., IL-17, IL-25), Th1 (IFN-γ), Th2 (e.g., IL-4,IL-13); the inhibition of Th1-, Th2- or TH17-specific transcriptionfactors; the inhibition of proliferation of effector T cells; theinduction of apoptosis of effector T cells; the induction of tolerogenicdendritic cell-specific genes, the induction of FoxP3 expression, theinhibition of IgE induction or IgE-mediated immune responses; theinhibition of antibody responses (e.g., antigen-specific antibodyproduction); the inhibition of T helper cell response; the production ofTGF-β and/or IL-10; the inhibition of effector function ofautoantibodies (e.g., inhibition in the depletion of cells, cell ortissue damage or complement activation); etc.

Any of the foregoing can be measured in vivo or may be measured invitro. One of ordinary skill in the art is familiar with such in vivo orin vitro measurements. Tolerogenic immune responses can be monitoredusing, for example, methods of assessing immune cell number and/orfunction, tetramer analysis, ELISPOT, flow cytometry-based analysis ofcytokine expression, cytokine secretion, cytokine expression profiling,gene expression profiling, protein expression profiling, analysis ofcell surface markers, PCR-based detection of immune cell receptor geneusage (see T. Clay et al., “Assays for Monitoring Cellular ImmuneResponse to Active Immunotherapy of Cancer” Clinical Cancer Research7:1127-1135 (2001)), etc. Tolerogenic immune responses may also bemonitored using, for example, methods of assessing protein levels inplasma or serum, immune cell proliferation and/or functional assays,etc. In some embodiments, tolerogenic immune responses can be monitoredby assessing the induction of FoxP3.

In some embodiments, the reduction of an undesired immune response orgeneration of a tolerogenic immune response may be assessed bydetermining clinical endpoints, clinical efficacy, clinical symptoms,disease biomarkers and/or clinical scores. Tolerogenic immune responsescan also be assessed with diagnostic tests to assess the presence orabsence of inhibitors.

In one embodiment, expression of the repaired FVIII results in theprevention, reduction, or elimination of inhibitors to a (r)FVIII. Thepresence of inhibitors can be assessed by determining one or moreantibody titers to the (r)FVIII using techniques known in the art andinclude Enzyme-linked Immunosorbent Assay (ELISA), inhibition liquidphase absorption assays (ILPAAs), rocket immunoelectrophoresis (RIE)assays, and line immunoelectrophoresis (LIE) assays.

In one embodiment, cells can be harvested, repaired, and then stored forfuture tolerance induction administration. The cells can be administeredin effective amounts, such as the effective amounts described elsewhereherein. Doses of dosage forms contain varying amounts of cellsexpressing a repaired FVIII, according to the invention. The amount ofexpressing cells present in the dosage forms can be varied according tothe nature and amount of the expressed FVIII, the therapeutic benefit tobe accomplished, and other such parameters. In embodiments, dose rangingstudies can be conducted to establish optimal therapeutic amount ofrepaired FVIII peptides to be expressed by the cells. In embodiments,the cells express a repaired FVIII in a dosage form in an amounteffective to generate a tolerogenic immune response to a (r)FVIIIepitope upon administration to a subject. It may be possible todetermine amounts of cellular expression effective to generate atolerogenic immune response using conventional dose ranging studies andtechniques in subjects. Dosage forms may be administered at a variety offrequencies. In one embodiment, at least one administration ofexpressing cells is sufficient to generate a pharmacologically relevantresponse. In one embodiment, at least two administrations, at leastthree administrations, or at least four administrations or more, of theexpressing cells are utilized to ensure a pharmacologically relevantresponse.

Prophylactic administration of the expressing cells can be initiatedprior to the onset of inhibitor development, or therapeuticadministration can be initiated after inhibitor development isestablished. In some embodiments, administration of cells expressingrepaired FVIII is undertaken e.g., prior to administration of the(r)FVIII. In exemplary embodiments, the expressing cells areadministered at one or more times including, but not limited to, 30, 25,20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 days priorto administration of the (r)FVIII. In addition or alternatively, thecells can be administered to a subject following administration of the(r)FVIII. In exemplary embodiments, cells are administered at one ormore times including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 25, 30, etc. days following administration of(r)FVIII.

In some embodiments, a maintenance dose is administered to a subjectafter an initial administration has resulted in a tolerogenic responsein the subject, for example to maintain the tolerogenic effect achievedafter the initial dose, to prevent an undesired immune reaction in thesubject, or to prevent the subject becoming a subject at risk ofexperiencing an undesired immune response or an undesired level of animmune response. In some embodiments, the maintenance dose is the samedose as the initial dose the subject received. In some embodiments, themaintenance dose is a lower dose than the initial dose. For example, insome embodiments, the maintenance dose is about ¾, about ⅔, about ½,about ⅓, about ¼, about ⅛, about 1/10, about 1/20, about 1/25, about1/50, about 1/100, about 1/1,000, about 1/10,000, about 1/100,000, orabout 1/1,000,000 (weight/weight) of the initial dose.

In some aspects, methods and compositions provided herein are useful inconjunction with established means of ITI for (r)FVIII. ITI protocolsfor hemophilia patients, including patients with high titer inhibitorsagainst (r)FVIII, are known in the art and are generally described,e.g., in Mariani et al., Thromb Haemost., 72: 155-158 (1994) andDiMichele et al., Thromb Haemost. Suppl 130 (1999). Administration ofthe repaired cells described herein can be conducted before, after,and/or concurrently with established ITI protocols and/or variationsthereof. For example, in some aspects, methods provide herein increasethe effectiveness of established ITI protocols (e.g., the degree and/orlikelihood of successful treatment) and/or reduce associated costs orside effects. In further aspects, methods provide herein allowestablished ITI protocols to be beneficially modified, e.g., to decreasethe frequency, duration, and/or dose of FVIII administration.

In a particular embodiment, the ITI strategy described herein iscombined with an antigenic peptide immunization strategy. In aparticular embodiment, the strategy described herein is combined withthe administration of overlapping sets of tolerogenic peptides based onamino acid differences between the (r)FVIII protein and the subject'sendogenous FVIII protein. In a particular embodiment, the strategydescribed herein is combined with the administration of overlapping setsof tolerogenic peptides based on amino acid differences between the(r)FVIII protein and the subject's repaired FVIII protein, described inU.S. Application 61/802,547, incorporated by reference herein. Theseamino acid residue differences between the subject's FVIII and (r)FVIIIcan be positioned or mapped within specific loci in the (r)FVIII,wherein the differing (r)FVIII amino acids—individually termed thereference locus—serves as a reference point or points for thepreparation of a set or sets of tolerizing peptides—termed tolerizingamino acids (“TAAs”)—that may incorporate T-cell epitopes capable ofinducing immune tolerance of, or the prevention, reduction, orelimination of inhibitor development to, the (r)FVIII. Each TAA within aset includes a (r)FVIII amino acid residing at a reference locus, and aTAA set includes between about 9 to 21 separate peptides of between 9 to21 amino acids in length, wherein the number of peptides in a TAA set isdirectly correlated with the amino acid length of the TAA (i.e., a TAAset containing TAAs 9 amino acids in length contains 9 peptides; a TAAset containing TAAs 10 amino acids in length contains 10 peptides,etc.).

A method of deriving TAA peptides is generally as follows. The firstpeptide of each TAA has as its first amino acid position the first aminoacid residue of a reference locus of the (r)FVIII, while the remainingamino acid residues are identical to the downstream amino acids in the(r)FVIII across the length of the TAA peptide. If only a single aminoacid residue difference exists at the locus (for example in the case ofa missense mutation or nsSNP), then the reference locus consists of asingle amino acid residue. If the differences encompass more than onecontiguous amino acid residue (for example in the case of somedeletions), then the first differing amino acid residue in the (r)FVIIIserves as the reference locus. For example, if the TAA is 9 amino acidsin length, the first amino acid in the first peptide is the first aminoacid of the reference locus, and the remaining 8 amino acid residues arethe 8 loci residues of the (r)FVIII immediately downstream from thereference locus (as determined from amino acid position 1 to 2332 in thewt FVIII protein). The second peptide of each TAA has as its secondamino acid position the reference locus, with the first amino acidposition being the first amino acid residue in the (r)FVIII immediatelyupstream from the reference locus, and the remaining 7 amino acidresidues being the 7 loci residues of the (r)FVIII immediatelydownstream from the reference locus. As such, for each successive TAApeptide in the TAA set, the reference locus is shifted one amino acidposition downstream, and the first amino acid reflects a shift from thepreceding peptide of one amino acid upstream in the (r)FVIII.Accordingly, the last TAA peptide of the set—in the preceding example,the ninth peptide—has the reference locus in the last amino acid residueposition, and be preceded by upstream amino acid residues—in thepreceding example, the 8 residues of the (r)FVIII immediately upstreamof the reference locus. The same method described above can be generallyused to create TAA sets of varying peptide sizes, wherein the referencelocus in each successive peptide in the set is shifted one positiondownstream and the first amino acid position in each successive peptideis shifted one residue upstream from the first amino acid position inthe preceding peptide, until the reference locus occupies the last aminoacid position in the last peptide of the set.

Following the method of generating sets of TAA as described above, a setof TAA peptides corresponds with a contiguous portion of the (r)FVIIIacross 2X−1 amino acids, where X is the length of the peptides containedin the set. For example, as described in the preceding example, a TAAset containing 9 peptides, each being 9 amino acids in length, will as aset overlap with 17 contiguous amino acids of the (r)FVIII. Furthermore,the contiguous (r)FVIII amino acid sequence overlapped by the TAApeptides includes X−1 amino acid residues upstream and X−1 amino acidresidues downstream from the first amino acid of the reference locuswithin the (r)FVIII, wherein X is the length of the peptides containedin the set. For example, a set of 9 peptides of 9 amino acids in lengthoverlaps with 8 amino acids upstream and 8 amino acids downstream fromthe first amino acid of the reference locus within the (r)FVIII. Thisgeneral process will be applicable to the generation of TAA sets formost identified amino acid differences, with a few exceptions, forexample in the derivation of TAA sets to a few BDD-rFVIIIrp syntheticlinker as described further herein.

In an alternative embodiment, the present strategy is combined withmethods of antigen specific muting of the B cell response to specificpotential antigens within the (r)FVIII. Accordingly, the repaired cellsare combined with a strategy using liposomal nanoparticles displayingpeptides comprising peptides derived from the (r)FVIII and based onamino-acid mismatches between the subject's endogenous FVIII protein, oralternatively the repair FVIII protein, and the (r)FVIII (as describedabove), and glycan ligands of the inhibitory co-receptor CD22. Methodsof producing antigenic liposomes displaying CD22 ligands are describedin, for example, Macauley et al., “Antigenic liposomes displaying CD22ligands induce antigen-specific B cell apoptosis,” J. Clin. Invest. 2013Jul. 1, 123(7): 3074-3083.

In some embodiments, the cells are associated with, combined with, oradministered with immunosuppressive compounds capable of inducingadaptive regulatory T cells. In one embodiment, the immunosuppresivecompounds may include, but is not limited to, IL-10, TGF-β, and/orrapamycin and/or other limus compounds, including but not limited tobiolimus A9, everolimus, tacrolimus, and zotarolimus, and/orcombinations thereof.

In one embodiment a “dose escalation” protocol can be followed, where aplurality of doses is given to the patient in ascending concentrations.Such an approach has been used, for example, for phospholipase A2peptides in immunotherapeutic applications against bee venom allergy(Müller et al. (1998) J. Allergy Clin Immunol. 101:747-754 and Akdis etal. (1998) J. Clin. Invest. 102:98-106).

In one aspect, the amount of cells to be administered can be determinedusing a stoichiometric calculation based on current ITI administrationprotocols. For example, the amount of cells to be administered can bebased on the equivalent quantity of the peptide that would beadministered in a standard ITI protocol which uses the full length(r)FVIII. To determine dosing period, the subject's dendritic cells'reactivity to the expressed repaired FVIII can be determined prior tothe start of cell administration, and then periodically monitored untiltolerance to the (r)FVIII is observed.

EXAMPLES Example 1: Ex Vivo Gene Repair

Examples are provided of an ex vivo gene repair strategies that can beperformed without the use of viral vectors. Genetic materials aredelivered to restore secretion of a wild-type full-length FVIII proteinto lymphoblastoid cells derived from a human HA patient with theintron-22 (I22)-inverted FVIII gene, designated F8_(I22I), usingelectroporation and TALE-nucleases (TALENs). A similar strategy can beused as an example to repair the naturally-occurring I22I-mutation incells from an animal model of HA (dogs of the HA canine colony locatedat the University of North Carolina in Chapel Hill). Canine (adipose)tissue, which can be induced to acquire many properties of hepatocytes,can be used.

Use of autologous cells is an attractive therapy for several reasons aslevels of blood clotting proteins needed to maintain hemostasis may bemore readily produced by expansion of large populations of cells ex vivoand reintroduction into the patient. Repair of the F8I22I gene residingin a B-lymphoblastoid cell-line derived from a patient with severe HAcaused by the I22I-mutation is effected by using electroporation todeliver (i) two distinct mRNAs encoding a highly specific heterodimericTALEN that targets a single human genome site located in F8 near the5′-end of I22 and (ii) the corresponding donor plasmid that carries the“editing cassette”, which is comprised of a functional 3′-intron splicesite ligated immediately 5′ of a partial F8 cDNA matched in sequencewith the wild-type sequence of exons 23-26 in the patient's ownI22-inverted F8 locus, flanked by “left” and “right” homology arms.

The use of viral-free methods to derive autologous cells of variousphenotypes and to stably introduce genetic information into the genomeis attractive. These methods can be effectively used to successfully“repair” the intron-22 (I22)-inverted human F8 locus (F8I22I), whicharises through a highly-recurrent mutational event essentiallyrestricted to the male germ-line. This same F8 abnormality, which iswidely known as the I22-inversion (I22I)-mutation, occurs naturally indogs, and results in spontaneous bleeding. Two large colonies of HA dogshave been established, one at the University of North Carolina in ChapelHill. Investigation of F8I22I at the molecular genetic, biochemical, andcellular levels to characterize its expression products have beenstudied in order to determine the immune response to replacement FVIII.Extensive sequencing efforts and analyses of the F8I22I and its mRNAtranscripts allow for an innovative gene repair strategy that exploitsnuclease technology, for example, transcription activator-like effector(TALE)-nuclease (TALEN) technology to repair the I22I-mutation.

Lymphoblastoid cells derived from HA patient with the I22I-mutation isobtained. The left (TALEN-L) and right (TALEN-R) monomers comprising theheterodimeric TALEN is shown in FIG. 3, which was specifically designedto cleave within the human F8 I22-sequence, ˜1 kb downstream of the3′-end of exon-22. In alternative embodiments, the TALENs targetsequences throughout the FVIII gene, with replacement of thecorresponding FV8 gene sequence on the donor sequence.

An example of a sequence that can be targeted includes a sequence withinintron 22 (tactatgggatgagttgcagatggcaagtaagacactggggagattaaat (SEQ. IDNo. 1)), where the underlined regions of sequence are recognized by theleft TAL Effector DNA-binding domain and the right TAL EffectorDNA-binding domain). Another example of a sequence that can be targetedincludes a sequence at the junction of exon 22 with intron 22(tggaaccttaatggtatgtaattagtcatttaaagggaatgcctgaata (SEQ. ID No. 2)),where the underlined regions of sequence are recognized by the left TALEffector DNA-binding domain and the right TAL Effector DNA-bindingdomain). Another example of a sequence that can be targeted withinintron 22 is depicted in FIG. 3(ttagtattatagtttctcagattatcaccagtgatactatggga (SEQ. ID No. 3)), wherethe underlined regions of sequence are recognized by the left TALEffector DNA-binding domain and the right TAL Effector DNA-bindingdomain). The two TALEN expression plasmids that target these sequences(or the mRNA) are co-transfected with the donor plasmid. The donorplasmid contains flanking homology regions to the intron 22 locus, whichallows for recombination of the donor plasmid into the chromosome. ThecDNA of exons 23 to 26 of the F8 gene is contained between the flankinghomology regions of the donor plasmid. The donor plasmid can alsocontain a suicide gene (such as the thymidine kinase gene from theherpes simplex virus), which allows counter-selection to avoid randomand multi-copy integration into the genome.

Electroporation (AMAXA Nucleofection system) and chemical transfection(with a commercial reagent optimized to this cell type) can be used astransfection methods for the lymphoblastoid cells. A plasmid containingthe green fluorescent protein (GFP) gene is introduced into the cellsusing both methods. The cells are analyzed by fluorescent microscopy toobtain an estimate of transfection efficiency, and the cells areobserved by ordinary light microscopy to determine the health of thetransfected cells. Any transfection method that gives a desirablebalance of high transfection efficiency and preservation of cell healthin the lymphoblastoid cells can be used. The TALEN mRNAs and the generepair donor plasmid is then introduced into the lymphoblastoid cellsusing a transfection method. The TALENs for the human lymphoblastoidcells and their target site are shown in FIG. 3.

Repair of the F8I22I in the adipose tissue-derived hepatocyte-like cellsfrom the I22I HA canine animal model is effected using electroporationto deliver mRNAs encoding an analogous TALEN that targets the 5′-end ofI22 in canine F8 and an analogous donor plasmid carrying a “splice-able”cDNA spanning canine F8 exons 23-26.

Adipose tissue is collected from these FVIII deficient dogs by standardliposuction. Stromal cells from the adipose tissue are reprogrammed intoinduced pluripotent stem cells (iPSC), as described by Sun et al.(“Feeder-free derivation of induced pluripotent stem cells from adulthuman adipose stem cells” Proc Natl Acad Sci USA. 106: 720-5, 2009) withtwo modifications: (i) mRNA of the reprogramming factors are used inplace of lentiviral vectors and (ii) the reprogramming is performedunder conditions of hypoxia, 5% O2, and in the presence of smallmolecules that have been found to increase the reprogramming efficiency.Once produced and characterized, pluripotent canine cells are obtained.

The defective FVIII sequence in iPSC is replaced by the correct sequenceusing site-specific TALE nucleases (see FIG. 4). The iPSC with repairedFactor VIII are differentiated into hepatocytes using well establishedprotocols (see, for example, Hay et al. “Direct differentiation of humanembryonic stem cells to hepatocyte-like cells exhibiting functionalactivities” Cloning Stem Cells. 9: 51-62, 2007; Si-Tayeb et al. “Highlyefficient generation of human hepatocyte-like cells from inducedpluripotent stem cells” Hepatology. 51: 297-305, 2010; and Cayo et al.“JD induced pluripotent stem cell-derived hepatocytes faithfullyrecapitulate the pathophysiology of familial hypercholesterolemia”Hepatology. May 31, 2012). In short, small colonies of iPSC are inducedto differentiate for the first 3 days into definitive endoderm bytreatment with 50 ng/mL Wnt3a and 100 ng/mL Activin A, and then into thehepatocyte lineage by 20 ng/mL BMP4. Two expression plasmids necessaryto produce mRNAs encoding a functional TALEN are obtained. These aredesigned to cleave and yield a double-stranded DNA break at only asingle site within the canine genome, located within canine F8 I22, ˜0.3kb downstream of the 3′-end of exon-22. The left (TALEN-L) and right(TALEN-R) monomers comprising this heterodimeric TALEN is shown above inFIG. 4.

A donor plasmid containing the sequence of the 3′-end of canine F8intron-22 and all of canine F8 exon-22 as the left homologous sequenceand the 5′-end of canine F8 intron-23 as the right homologous sequenceto provide an adequate length of genomic DNA for efficient homologousrecombination at the target site (i.e., the TALEN cut site) is created.The TALEN mRNAs and the gene repair donor plasmid are introduced intothe pluripotent canine cells using a transfection method describedherein.

Likewise, in humans, human iPSCs are electroporated with the human F8TALENs & donor plasmid described above, to assess candidategenome-editing tools (which were designed to be equally capable of“editing” the I22-sequence in the wild-type and I22-inverted F8 loci, F8and F8I22I, respectively) for their efficiency of site-specific generepair. The genomic DNA at the repaired F8 loci, as well as the mRNAsand expression products synthesized by, the cells described above areassessed before and after electroporation.

The TALEN gene repair method described above inserts F8 exons 23-26immediately downstream (telomeric) to F8 exons 1-22 to encode a FVIIIprotein. Genomic DNA, spliced mRNA, and protein sequences differ amongnormal, repaired, and unrepaired cells (see FIG. 5). Gene repair isverified in genomic DNA through the use of PCR. Specific PCR primers aredesigned to amplify across the homologous recombination target sequencein unrepaired and repaired cells. A common primer is placed toward theend of exon-22. An I22I-specific primer is placed in the sequencetelomeric to exon-22 in the I22I-inverted cells. A Repaired-specificprimer is placed in the inserted exon 23-26 sequence. Primer design isshown in FIG. 8. Separate sets of primers are designed for human andcanine sequences.

Characterization of the genomic DNA at the repaired F8 loci, as well asthe mRNAs and expression products synthesized by, the cells describedabove, before and after electroporation are performed.

A quantitative RT-PCR test that specifically detects and quantifies themRNA transcripts from normal and I22I cells is used. The quantitativeRT-PCR test uses three separate primer sets: one set to detect exons1-22, one set to detect exons 23-26, and one set that spans theexon-22/exon-23 junction. mRNA is purified from cells before and aftertransfection. The existing primer design to probe mRNA from the humancells is used. Primers against canine sequences are designed using thesame strategy and then the mRNA from the canine cells is probed usingthese new primers. An increased signal from the exon-22/exon-23 junctionreaction in repaired cells, relative to unrepaired cells should beobserved.

Monoclonal antibody ESH8, which is specific for the C2-domain of theFVIII protein, is be used. NIH3T3 cells were transfected with expressionconstructs encoding full-length and I22I F8 genes and then assayed byflow cytometry. Signal from the ESH8 antibody was high in cellstransfected with the full-length construct but virtually absent in cellstransfected with the I22I construct. The ESH8 antibody is used to testtransfected cells. There should be an increased signal in repaired cellsrelative to unrepaired cells. Secreted FVIII levels, as measured byELISA, are dramatically lower in I22I cells relative to normal cells.Whole-cell lysates and supernates from transfected cells are obtainedand tested for FVIII concentration by ELISA. There should be an increasein FVIII concentration in the supernates from repaired cells relative tounrepaired cells.

In another example, canine blood outgrowth endothelial cells (cBOECs)and canine iPSCs derived from canine adipose tissue can be transfectedwith TALENs that target the F8I22I canine gene and a plasmid repairvehicle that carries exons 23-26 of cF8. TALENs are expected to makeDSBs in the F8I22I DNA at the target site to allow “homologousrecombination and repair” of the canine F8 I22I gene by insertion ofexons 23-26 of the canine F8. The TALENS are designed to cleave andyield a DSB at only a single site within the canine genome, locatedwithin canine F8 I22, (˜0.3 kb) downstream of the 3′-end of exon-22. Thedonor plasmid contains the sequence of canine F8 exons 23-26 flanked bythe 3′-end of canine F8 intron-22 and all of canine F8 exon-22 as theleft homologous sequence and the 5′-end of canine F8 intron-23 as theright homologous sequence to provide an adequate length of genomic DNAfor efficient homologous recombination at the target site.

Feasibility of deriving canine iPSCs is well established. An mRNAtranscript that enables expression of the so called “Yamanaka” genescoding for transcription factors OCT4, SOX2, KLF4 and C-MYC to induceiPSCs from canine adipose derived stem cells (hADSCs). iPSCs have beentransfected using Nucleofector. For transfection, Qiagen's Polyfecttransfection reagents can be used with TALENs for many cell types,including BOECs. Transfection methods can be assessed using commercialreagents and transfected cells can be analyzed by fluorescent microscopyto obtain an estimate of transfection efficiency, while viability can bedetermined by Trypan Blue dye exclusion. The transfection method thatgives the best balance of high transfection efficiency and preservationof cell health can be used.

Prior to commencing transfection with the TALENS and repair plasmid, thecleavage activity of the TALENs against the target site can be analyzed.This can be done by monitoring TALEN induced mutagenesis (Non-HomologousEnd Joining Repair) via a T7 Endonuclease assay. To assess potentialrisk of unintended genomic modification induced by the selected repairmethod, off-site activity is analyzed following transfection. In silicoidentification based on homologous regions within the genome can be usedto identify the top 20 alternative target sites containing up to twomismatches per target half-site. PCR primers can be synthesized for thetop 20 alternative sites and Surveyor Nuclease (Cel-I) assays(Transgenomics, Inc.) can be performed for each potential off-targetsite.

Transfection for expression and secretion of FVIII can be assessed inthe various cell types before and after transfection. Genomic DNA isisolated from cells before and after transfection. Purified genomic DNAis used as template for PCR. Primers are designed for amplification froma FVIII I22I-specific primer only in unrepaired cells, and amplificationfrom the repaired-specific primer only in repaired cells. RT-PCR canspecifically detect and quantify the mRNA hF8 transcripts from normaland I22I cells. The quantitative RT-PCR test uses three separate primersets: one set to detect exons 1-22, one set to detect exons 23-26, andone set that spans the exon-22/exon-23 junction. mRNA is purified fromcells before and after transfection, with an increased signal from theexon-22/exon-23 junction reaction in repaired cells, relative tounrepaired cells. Flow-cytometry based assays may also be used for FVIIIprotein in peripheral blood mononuclear cells (PBMCs).

iPSCs derived from canine adipose tissue engineered can be conditionedto secrete FVIII to hepatocyte-like tissue. Canine iPSCs are conditionedtoward hepatocyte like cells using a three step protocol as described byChen et al. that incorporates hepatocyte growth factor (HGF) in theendodermal induction step (Chen Y F, Tseng C Y, Wang H W, Kuo H C, YangV W, Lee O K. Rapid generation of mature hepatocyte-like cells fromhuman induced pluripotent stem cells by an efficient three-stepprotocol. Hepatology. 2012 April; 55(4):1193-203).

Subpopulations of cBOECs are segregated and expanded and thencharacterized for the expression of endothelial markers, such as MatrixMetalloproteinases (MMPs), and cell-adhesion molecules (JAM-B, JAM-C,Claudin 3, and Claudin 5) using RT-PCR. Detailed RT-PCR methods,including primers for detecting expression of mRNA transcripts of thecell-adhesion molecules of interest and detailed immunohistochemistrymethods to detect the proteins of interest, including a list of highaffinity antibodies have been published by Geraud et al. (Géraud C, etal. Unique cell type-specific junctional complexes in vascularendothelium of human and rat liver sinusoids. PLoS One. 2012;7(4):e34206). Antibodies that detect JAM-B, JAM-C, Claudin 3, andClaudin 5 may be purchased from LifeSpan Biosciences (www.lsbio.com).

One subpopulation of co-cultured cBOECs can be prepared and segregatedearly (before ˜4 passages of outgrowth). Later segregation of thesubpopulation can occur after ˜10 passages. After 1 week of co-culture,two cBOECs subpopulations can be compared for expression and secretionof FVIII, and suitability for engraftment in the canine liver.Co-culturing of hepatocytes can be done with several cell typesincluding human umbilical vein endothelial cells (HUVECs). cBOECs can beused as surrogates for HUVECS in this system. Once the repaired cBOECs(with the repaired FVIII gene) are obtained, the cells can be used toinduce immune tolerance in canines with high titer-antibodies to FVIII.

Example 2: Protocol for Factor VIII Gene Repair in Humans

Obtaining a Blood Sample

A protocol for gene repair of the F8 gene in blood outgrowth endothelialcells (BOECs) is described in the following example. First, a bloodsample is obtained, with 50-100 mL of patient blood samples obtained byvenipuncture and collection into commercially-available, medical-gradecollecting devices that contain anticoagulants reagents, followingstandard medical guidelines for phlebotomy. Anticoagulant reagents thatare used include heparin, sodium citrate, and/orethylenediaminetetraacetic acid (EDTA). Following blood collection, allsteps proceed with standard clinical practices for aseptic technique.

Isolating Appropriate Cell Populations from Blood Sample

Procedures for isolating and growing blood outgrowth endothelial cells(BOECs) have been described in detail by Hebbel and colleagues (Lin, Y.,Weisdorf, D. J., Solovey, A. & Hebbel, R. P. Origins of circulatingendothelial cells and endothelial outgrowth from blood. J Clin Invest105, 71-77 (2000)). Peripheral blood mononuclear cells (PBMCs) arepurified from whole blood samples by differential centrifugation usingdensity media-based separation reagents. Examples of such separationreagents include Histopaque-1077, Ficoll-Paque, Ficoll-Hypaque, andPercoll. From these PBMCs multiple cell populations can be isolated,including BOECs. PBMCs are resuspended in EGM-2 medium without furthercell subpopulation enrichment procedures and placed into 1 well of a6-well plate coated with type I collagen. This mixture is incubated at37° C. in a humidified environment with 5% CO2. Culture medium ischanged daily. After 24 hours, unattached cells and debris are removedby washing with medium. This procedure leaves about 20 attachedendothelial cells plus 100-200 other mononuclear cells. Thesenon-endothelial mononuclear cells die within the first 2-3 weeks ofculture.

Cell Culture for Growing Target Cell Population

BOECs cells are established in culture for 4 weeks with daily mediumchanges but with no passaging. The first passaging occurs at 4 weeks,after approximately a 100-fold expansion. In the next step, 0.025%trypsin is used for passaging cells and tissue culture plates coatedwith collagen-I as substrate. Following this initial 4-weekestablishment of the cells in culture, the BOECs are passaged again 4days later (day 32) and 4 days after that (day 36), after which time thecells should number 1 million cells or more.

In Vitro Gene Repair

In order to affect gene repair in BOECs, cells are transfected with0.1-10 micrograms per million cells of each plasmid encoding left andright TALENs and 0.1-10 micrograms per million cells of the repairvehicle plasmid. Transfection is done by electroporation,liposome-mediated transfection, polycation-mediated transfection,commercially available proprietary reagents for transfection, or othertransfection methods using standard protocols. Following transfection,BOECs are cultured as described above for three days.

Selection of Gene-Repaired Clones

Using the method of limiting serial dilution, the BOECs are dispensedinto clonal subcultures, and grown as described above. Cells areexamined daily to determine which subcultures contain single clones.Upon growth of the subcultures to a density of >100 cells persubculture, the cells are trypsinized, re-suspended in medium, and a1/10 volume of the cells is used for colony PCR. The remaining 9/10 ofthe cells are returned to culture. Using primers that detectproductively repaired F8 genes, each 1/10 volume of colonies arescreened by PCR for productive gene repair. Colonies that exhibitproductive gene repair are further cultured to increase cell numbers.Using the top 20 predicted potential off-site targets of the TALENs,each of the colonies selected for further culturing is screened forpossible deleterious off-site mutations. The colonies exhibiting theleast number of off-site mutations are chosen for further culturing.

Preparation of Cells for Re-Introduction into Patients by Conditioningand/or Outgrowth

Prior to re-introducing the cells into patients, the BOECs are grown inculture to increase the cell numbers. In addition to continuing cellculture in the manner described above, other methods can be used tocondition the cells to increase the likelihood of successful engraftmentof the BOECs in the liver sinusoidal bed of the recipient patient. Theseother methods include: 1) co-culturing the BOECs in direct contact withhepatocytes, wherein the hepatocytes are either autologouspatient-derived cells, or cells from another donor; 2) co-culturing theBOECs in conditioned medium taken from separate cultures of hepatocytes,wherein the hepatocytes that yield this conditioned medium are eitherautologous patient-derived cells, or cells from another donor; or 3)culturing the BOECs as spheroids in the absence of other cell types.

Co-culturing endothelial cells with hepatocytes is described further inthe primary scientific literature (e.g. Kim, Y. & Rajagopalan, P. 3Dhepatic cultures simultaneously maintain primary hepatocyte and liversinusoidal endothelial cell phenotypes. PLoS ONE 5, e15456 (2010)).Culturing endothelial cells as spheroids is also described in thescientific literature (e.g. Korff, T. & Augustin, H. G. Tensional forcesin fibrillar extracellular matrices control directional capillarysprouting. J Cell Sci 112 (Pt 19), 3249-3258 (1999)). Upon growing thecolonies of cells to a total cell number of at least 1 billion cells,the number of cells needed for injection (>50 million cells) into thepatient are separated from the remainder of the cells and used in thefollowing step for injection into patients. The remainder of the cellsare aliqouted and banked using standard cell banking procedures.

Injection of Gene-Repaired BOECs into Patients

BOECs that have been chosen for injection into patients are resuspendedin sterile saline at a dose and concentration that is appropriate forthe weight and age of the patient. Injection of the cell sample isperformed in either the portal vein or other intravenous route of thepatient, using standard clinical practices for intravenous injection.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application, to the extent allowedby law.

A number of aspects have been described. Nevertheless, it will beunderstood that various modifications may be made. Furthermore, when onecharacteristic or step is described it can be combined with any othercharacteristic or step herein even if the combination is not explicitlystated. Accordingly, other aspects are within the scope of the claims.

What is claimed is:
 1. An in vitro or ex vivo method of repairing amutated F8 gene, the method comprising: (i) providing an endothelialcell comprising an inversion mutation in the F8 gene; (ii) introducinginto the cell an isolated nucleic acid encoding a nuclease that targetsan inversion mutation of the F8 gene and creates a double stranded breakin the mutated F8 gene; and a donor sequence comprising (a) a nucleicacid encoding a truncated FVIII polypeptide or (b) a native F8 3′ spliceacceptor site operably linked to a nucleic acid encoding a truncatedFVIII polypeptide, wherein the donor sequence is flanked by nucleic acidsequences homologous to the nucleic acid sequences upstream anddownstream of the double stranded break in the F8 gene and (iii)obtaining an endothelial cell that comprises a repaired inversionmutation in the F8 gene.
 2. The method of claim 1 wherein theendothelial cell that comprises a repaired inversion mutation in the F8gene is administered to a subject.
 3. The method of claim 1, wherein thenuclease is a zinc finger nuclease (ZFN), Transcription Activator-LikeEffector Nuclease (TALEN), or a CRISPR (Clustered Regularly InterspacedShort Palindromic Repeats)-associated (Cas) nuclease.
 4. The method ofclaim 1, wherein the nuclease targets intron 22 or the exon 22/intron 22junction of the F8 gene.
 5. The method of claim 1, wherein the nucleasetargets intron 1 or the exon 1/intron 1 junction of the F8 gene.
 6. Themethod of claim 1, wherein the mutation in the F8 gene is an intron 22inversion.
 7. The method of claim 1, wherein the cells are bloodoutgrowth endothelial cells (BOECs) or liver sinusoidal endothelialcells (LSECs).
 8. The method of claim 7, wherein the blood outgrowthendothelial cells (BOECs) have been co-cultured with hepatocytes orliver sinusoidal endothelial cell (LSECs), or both.
 9. The method ofclaim 7, wherein the blood outgrowth endothelial cells (BOECs) have beenco-cultured with induced pluripotent stem cells (iPSCs).
 10. The methodof claim 2, wherein the endothelial cell that comprises a repairedinversion mutation in the F8 gene confers an improved coagulationfunctionality of the encoded FVIII protein of the subject compared tothe coagulation functionality of the FVIII protein encoded by themutated F8 gene of the subject.
 11. The method of claim 2, wherein thesubject has hemophilia A.
 12. The method of claim 2, wherein prior toadministering the endothelial cell that comprises a repaired inversionmutation in the F8 gene to the subject, the repaired endothelial cell isisolated out of a population of endothelial cells that do not have arepaired F8 gene.
 13. The method of claim 12, wherein prior toadministering the endothelial cell that comprises a repaired inversionmutation in the F8 gene to the subject the endothelial cell thatcomprises a repaired inversion mutation in the F8 gene is expanded invitro to produce a population of endothelial cells that comprise arepaired inversion mutation in the F8 gene.
 14. The method of claim 13,wherein the endothelial cells that comprise a repaired inversionmutation in the F8 gene are blood outgrowth endothelial cells (BOECs) orliver sinusoidal endothelial cells (LSECs).
 15. The method of claim 14,wherein the blood outgrowth endothelial cells (BOECs) (i) have beenco-cultured with hepatocytes or liver sinusoidal endothelial cell(LSECs), or both; (ii) have been cultured in growth medium supplementedwith conditioned medium from hepatocytes; or (iii) have been co-culturedwith induced pluripotent stem cells (iPSCs).
 16. The method of claim 15,wherein the subject has hemophilia A.
 17. The method of claim 11,wherein the administration of the endothelial cell that comprises arepaired inversion mutation in the F8 gene induces a tolerogenic immuneresponse to a replacement FVIII protein.
 18. The method of claim 16,wherein the administration of the endothelial cells that comprise arepaired inversion mutation in the F8 gene induces a tolerogenic immuneresponse to a replacement FVIII protein.
 19. The method of claim 7,wherein the blood outgrowth endothelial cells (BOECs) have been culturedin growth medium supplemented with conditioned medium from hepatocytes.